Major royal jelly proteins accelerate onset of puberty and promote ovarian follicular development in immature female mice

Xin Liu, Chenmin Jing, Yong Chen, Fngxiong Shi, Choqing Li, Lirong Shen,?

aDepartment of Food Science and Nutrition, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University,Hangzhou, China

bCollege of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China

cUSDA ARS, JM-USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA, 02111, United States

ABSTRACT

The major royal jelly proteins (MRJPs) are the central constituents responsible for the specific activities of royal jelly. Here MRJPs via oral administration daily for 45 consecutive days were evaluated the effects on the reproductive parameters in immature female mice (FM). Neonatal FM were divided into four groups fed MRJPs with doses of 0, 125, 250 and 500 mg/kg/body weight (M125, M250 and M500). The results in M125, M250 and M500 showed that the times of estrus were accelerated by 10.7%, 15.5% and 10.7%, the secondary follicles number were increased by 50.7%, 78.8% and 38.6%, the Graafian follicles were increased by 600.0% and 774.0% and 150.0%, respectively. M500 induced multi-oocyte follicles. The serum estradiol levels of the three groups were increased by 47.1%, 64.9% and 31.1%, the action of MRJPs raising hormone secretion level is mainly via upregulating expression of ERβ gene. Antioxidant parameters of ovarian tissue showed that the malondialdehyde levels in M125 and M250 were decreased, the superoxide dismutase activities and glutathione peroxidase activities in M125 and M250 were increased.In conclusion, MRJPs may accelerate onset of puberty and promote follicular development in FM. Our findings would facilitate better understanding of the benefit effect of MRJPs as the key ingredient in royal jelly on promoting fertility performance.

Keywords:

Major royal jelly proteins

Immature female mice

Puberty onset

Follicular development

Action mechanism

1. Introduction

Puberty is a crucial stage in reproductive maturation, especially among females, during which the sex hormones secrete and the gonads mature. The onset of puberty in females is highly sensitive to the nutritional status and the amount of energy reserves of the organism [1]. Royal jelly (RJ), a creamy pulpy substance secreted from the hypopharyngeal and mandibular glands of nurse honeybees (Apis mellifera L.), is the exclusive food source of the queen bees and the larvae of worker bees during the first 3 days after emergence [2]. It possess numerous pharmacological and nutritional properties, such as antifatigue [3], antibacterial [4], antioxidation [5], anti-aging [6], lifespan extension [7], anti-inflammatory[8], antitumor [9], enhancing immune activity [10] and improving hyperglycemia [11]. Indeed, RJ has been used worldwide for many years as medical products and functional foods [12].

RJ were composed of proteins, sugars, lipids, vitamins and free amino acids, etc [13]. Moreover, proteins are the major components of RJ accounting for nearly 50% of its dry matter. Analyses of the RJ proteins show that 82%–90% (m/m) is constituted by the major RJ proteins (MRJPs) [14]. The MRJP family belong glycosylated protein composed of nine members with molecular weight of 49–87 kD is the major factor responsible for the physiological activities of RJ [15]. MRJP1 (royalactin) was proved to be the key factor of caste differentiation in the honeybee [16], as well as appears to possess growth-factor-like activity, including promoting DNA synthesis, proliferation, and suppression of apoptosis in rat hepatocytes [17]. The other MRJPs also play important roles in determining the fate of the developing larvae in honeybee [2,18] working on Drosophila melanogaster reported that not only MRJP1 but all the MRJPs together Influence and enhance the longevity of Drosophila[2]. Buttstedt et al. [19] observed that there is no single factor for queen differentiation like MRJP1. They suggested that the growth is driven by the amount of foods ingested while MRJP1 provides essential amount of nutrients for the queen larvae.

Estrogen plays an important role in the growth, differentiation and function of many targets including the female and male reproductive system through estrogen receptor (ER). Several previous studies indicated that RJ can exhibit weak estrogen-like effects [20]. Three fatty acids, 10-hydroxy-trans-2-decenoic acid(10- HDA), 10-hydroxydecanoic acid and trans-2-decenoic acid,and a sterol, 24-methylenecholesterol isolated from RJ exhibited estrogenic activity in immature rats [21]. Oral and intramuscular administration of RJ improves estrus response of female Awassi ewes model [22,23]. RJ could also be effective in ameliorating pregnancy rates and the lambing rates. Furthermore, RJ promotes ovarian follicular development and increases steroid hormones in immature rats. The effect of RJ on in vitro maturation of ovine oocyte has been investigated. RJ treatment caused a significant increase in oocyte and nuclear maturation rate, fertilization rate and blastocyst formation [24]. MRJP1 induce larval develop into queen bee and increase body size and ovarian development in honeybees, which suggests that the protein has estrogen-like effects [16]. A recent randomized controlled clinical trial that was done on 90 married postmenopausal women 50 to 65-year-old expressed that vaginal RJ is considerably more effective than conjugated estrogens and lubricant in the improvement of quality of life, sexual and urinary function in postmenopausal women [25].

The estrogenic effect of RJ is not due to the sex hormones of RJ itself, but its active ingredients [16,22]. In recent years, we explored a new technique to preparing freeze-dried MRJPs powder with fresh RJ as raw materials by using ultra filtration equipment in order to preserve the activity of fresh RJ. The freeze-dried MRJPs have retained its biological function of RJ and remains stable under room temperature [26,27]. Our studies have demonstrated that MRJPs possesses functions to increase lifespan, feeding and fecundity in Drosophila [2]. However, the mechanism of action of MRJPs on reproductive function in mammalians remains unclear.

Previous data prompted us to study the effect of MRJPs on puberty and fertility parameters in immature female mice. MRJPs may exert reproductive function by altering hormonal secretions or by containing hormone-like compounds. Therefore, the aim of the present study was to evaluate the effects of MRJPs on puberty onset and ovarian follicular development in immature female mice.

2. Materials and methods

2.1. Reagents

Radioimmunoassay kits to assay estradiol (E2), and progesterone (P) were purchased from Xinfan Bioengineering Corporation(Shanghai, China). Malonaldehyde (MDA) kit (cat.A003-1), SOD kit(cat.A001-3), glutathione peroxidase (GSH-Px) kit (cat. A005) and Bradford kit (cat. A045-2) were produced by Nanjing Jiancheng Bioengineering Company (Nanjing, China).

2.2. Preparation and analysis of the freeze-dried MRJPs powder

The freeze-dried MRJPs powder was prepared with fresh RJ by using ultra filtration equipment [26], and stored at ?80°C until use.The concentrations of total protein, total sugar, 10-HDA, water and ash were determined according the National Food Safety Standard GB9697-2008 [28]. The presence of proteins of the MRJPs powder was analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the standard method [29].

2.3. Animals and treatments

Immature female ICR mice were obtained from the Animal Center of Nanjing Agricultural University (Nanjing, China). Animals were maintained with a 14/10 h light/dark cycles and freely supplied with standard food and water. The experiments were conducted with the approval of the Ethical Committee for Animal Experiments at Nanjing Agriculture University. Neonatal mice (postnatal day [PND] 1) were randomly divided into three experimental groups based on their body weight. They received different dose of freeze-dried MRJPs powder: 125 mg/kg·body weight(M125), 250 mg/kg·body weight (M250) and 500 mg/kg·body weight (M500) by oral route, respectively. The control mice (Control blank, CK) were fed equal volumes of normal saline in the same manner. These doses were set according to previous studies [2]. MRJPs powder diluted in saline solution was administrated daily for 45 consecutive days. All mice were weighed every 3 days throughout the experimental period.

2.4. Calculation of ovary index and uterus index

Animals were sacrificed after 24 h of MRJPs administration for 45 days. Their ovaries and uteruses (bilateral) were collected carefully and the fascia and adipose tissue was removed from them. The wet weight of the ovaries and uteruses were measured,respectively. The ovary index (OI) was calculated according to the following formula: OI = ovarian wet weight (mg)/body weight (g)× 100%. The uterus index (UI) was calculated according to the following formula: UI = uterine wet weight (mg)/body weight (g) ×100%.

2.5. Vaginal opening and estrous cyclicity

Female mice treated orally with MRJPs were weaned at 21 days of age, housed four per cage, and followed daily for vaginal opening.Vaginal cytology was assessed daily between 9:00 AM and 10:00 AM before and until the end of the treatment to determine the estrous cycle of each mouse. Vaginal cells were collected via a sterile cotton swab saturated with normal saline (NaCl, 0.9%), and then placed on a clean glass slide to form well-proportioned tracks of smears. The smears were stained with Giemsa and examined under light microscope. Stages of estrous cycle were determined based on vaginal cytology, as previously described [30].

2.6. Ovarian histology evaluation and follicle classification

The ovaries and uteri were dissected and cleaned from fatty tissues and were weighed afterwards. One of the two ovaries from each mouse were fixed in 10% formalin, sectioned at 6 μm thickness and stained with hematoxylin and eosin (HE) for histological analysis. The five largest sections of each ovary were used for follicular investigation. Histopathological examination was performed to measure the effects of MRJPs on the mean number of primordial follicles (PF I), primary follicles (PF II), secondary follicles (SF), Graffian follicles (GF). Samples were evaluated for histological changes under an optical microscope and images were captured for further analysis by using an image processing software [31].

2.7. Antioxidation assay of ovarian tissues

The other one of the two ovaries from each mouse were maintained at ?80°C for future analysis. A portion of ovarian tissues for all the assays were homogenized in a 0.9% NaCl solution (NaCl/ovarian tissues, 9:1, V/V). Tissue homogenates were centrifuged for 15 min at 15000 × g, and then the clear upper supernatants were removed for analysis. Analysis of the samples was carried out after the end of the experiment. MDA levels, SOD activity, GSH-Px activity and protein content were measured separately with the appropriate test kits. The protocols used were provided by the manufacturers. The protocols used were provided by the manufacturers. The level of lipidperoxidation was expressed as mmol MDA per mg of protein. A single unit of SOD and GSH-Px activity was defined as the amount of enzyme required to cause a 50% inhibition and expressed as enzyme units per gram of the total protein.For each parameter measured, at least three replicates were used for each control or treatment group.

2.8. Hormonal assay in serum

Blood collection of enucleation were performed after treatment.Then they were kept at room temperature for 2 h and centrifuged at 3000 r/min for 10 min. Subsequently, serum was transferred into 1.5 mL polypropylene tubes, and stored at ?20°C. The levels of E2and P in the collected serum were determined by radioimmunoassay kit. The protocols used were provided by the manufacturers.

2.9. Quantitative RT-PCR analysis of hormone receptor gene from ovary tissue

Samples of ovary tissue were collected and frozen in liquid nitrogen and stored at ?80°C for analysis of mRNA levels.Total RNA was extracted from each sample using RNAiso Plus(TaKaRa) and then was synthesized into cDNA with a PrimeScrit RT reagent Kit (TaKaRa) according to the manufacturer’s protocol.β-actin, Estrogen Receptor α (ERα), Estrogen Receptor β (ERβ), were selected for quantitative RT-PCR analysis. The mRNA expression of β-actin gene was used as an internal control. The following primers were used: ERα: 5′-CAAGGTAAATGTGTGGAAGGCA-3′(forward) and 5′-ATGGGAAAGAAT GAGAAGGAGC-3′(reverse);ERβ: 5′-GACGAAGAGTGCTGTCCCAA-3′(forward) and 5′-TCAGCTTCCGGCTACTCTCT-3′(reverse). All primers were designed according to previous report [32] and synthesized by TaKaRa.The ViiATM7 Real-Time PCR system with fast 96-well block and SYBR?Premix Ex TaqTMII (TliRNase H Plus) (TaKaRa) were used in the quantitative RT-PCR analysis. The comparative-cycle-threshold(Ct) method was used to analyze the data. Levels of gene expression in all groups were expressed as a ratio of the N25group value. Data were analyzed by using the 2–ΔΔCtmethod, which is a convenient way to analyze the relative changes in gene expression [33]. All experiments were repeated three times. For all reactions, melting analysis con firmed that only a single amplicon and the reference gene (β-actin) were generated [32].

2.10. Statistical analysis

Data analysis was performed using the software, Statistical Product and Service Solutions (SPSS) 22.0. The data from biological assays were presented as mean ± SD (standard deviation). The statistical differences between the values were shown by One-way analysis of variance (ANOVA) test. If there was a significant difference (P < 0.05) between groups, Duncan analysis was used to compare all the groups in pairs, and P < 0.05 was considered significantly different.

3. Results

3.1. Analysis of freeze-dried MRJPs

The freeze-dried MRJPs powder separated from fresh RJ by using ultra filtration equipment was shown in Fig. 1A. The powder contains total proteins, total sugar, 10-HDA, water and ash with 62.5%,23.6%, 1.49%, 4.98% and 1.42%, respectively. It is estimated that the content of MRJPs, the glycoproteins with N-linked glycosylation sites were composed of proteins and sugars was more than 80%.The SDS-PAGE analysis of the MRJPs powder showed more than 4 bands, MRJP1, MRJP2, MRJP3 and MRJP5, with 25–87 kDa (Fig. 1B),which were reported previously [34]

Fig. 1. The freeze-dried MRJPs powder prepared with fresh RJ using ultra filtration equipment (A), and SDS-PAGE analysis of MRJPs (B). Lanes: M, protein marker; MRJPs,soluble RJ proteins.

3.2. Effects of MRJPs on body weights

The effects of oral administration of MRJPs on the body weight(BW) of neonatal female mice during 45 days were observed(Fig. 2A). On the 1st day, there was not significant difference among all groups in the mean BW (P = 0.104). In general, the BW in all groups were increased with growth. The BW of female mice in M125, M250 and M500 at the 45th days were 27.96, 30.40 and 26.66 g, which were increased by 17.0%, 27.3% and 11.6% (P < 0.05)compared to CK (23.89 g), respectively (Fig. 2B). The results suggest that oral administration of MRJPs to neonatal females caused a sustained increase in BW gain.

Fig. 2. (A) Changes in BW after MRJPs treatment in female mice. Early prepubertal supplement with MRJPs (125, 250 and 500 mg/kg) significantly increased BW (P <0.05). (B) Effect of supplemental MRJPs on final BW of ean ± SE in each group (n = 6). Different lowercase letters (a–d) above bars indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range tests.

3.3. Effects of MRJPs on ovary index and uterus index

The effects of supplemental MRJPs on OI and UI of female mice at the 45th day are presented in Fig. 3A and 3B. The OI in M125, M250 and M500 were 0.71, 0.74 and 0.57 mg/g, which were increased by 26.8% (P = 0.001), 32.1% (P = 0.000127) and 1.7% (P = 0.991)compared to CK (0.56 mg/g), respectively. The OI in M250 with significant increase in comparison to that of CK acted as the better MRJPs dose.

Fig. 3. Effects of neonatal treatment with MRJPs on the ovarian index (OI) and uterine index (UI). (A) Histogram of OI and (B) Histogram of OI. Data are expressed as mean ± SE (n = 6). Different lowercase letters (a–b) above bars indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range tests.

The UI in M150, M250 and M500 were 2.94, 3.19 and 2.89 mg/g,which were increased by 4.3% (P = 0.641), 13.1% (P = 0.010) and 2.5% (P = 0.908) compared to CK (2.82 mg/g), respectively. The UI in M250 with significant increase in comparison to CK acted as the best MRJPs.

3.4. Effects of MRJPs on puberty onset

To determine the onset of puberty, the vaginal opening (VO)time was checked from postpartum day (PND) 24 to PND 35 (Fig. 4).The first VO occurred on PND 26 in M250 group. The days of the VO were identified as 28.3, 26.8 and 28.3 days in M125, M250 and M500, which were accelerated by 10.7%, 15.5% and 10.7% (P < 0.01)compared to CK (31.7 days), respectively. The results suggest that supplemental MRJPs can accelerate onset of puberty in female mice.

Fig. 4. Effect of supplemental MRJPs on the puberty onset of female mice.The vaginal opening times of all MRJPs treatment groups were significantly faster than the CK group (n = 6 per group). Different lowercase letters (a–b) above bars indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range tests.

3.5. Effects of supplemental MRJPs on the estrous cycle

The effects of supplemental MRJPs on the estrous cycle were shown in Table 1. The control mice exhibited regular cycle and normal duration of each phase of estrous cycle. No significant changes in the estrous cycle length, as well as in the duration of the proestrus, metestrus and diestrus phases was observed in all MRJPs treatment groups except M250 compared to CK (Table 1).The treatments with M250 (1.90 days) caused a significant increase in the length of the estrous phase compared to controls (1.66 days,P = 0.046).

Table 1Effect of MRJPs on the length of various phases in the estrous cycle.

Table 2Effects of supplemental MRJPs on antioxidant properties.

3.6. Effects of MRJPs on ovarian follicles

The typical morphology in the mouse ovary as visualized after HE staining is shown in Fig. 5. Morphological evaluation was performed to compare the structures of PFI, PFII, SF and GF in mouse ovarian sections from all groups. The integrity of the ovaries was well preserved in all MRJPs-treated groups, which were similar to that in CK. Morphologically normal follicles were characterized by a round or oval oocyte, presenting a well-delimited nucleus with uncondensed chromatin, surrounded by healthy granulosa cells closely juxtaposed to the oocyte. Interestingly, multi-oocyte follicles (MOFs), namely a single follicle with two or more oocytes were observed in the treatment of high dose MRJPs (Fig. 5D).

Fig. 5. Representative light microscopy images of ovarian issues in immature FM. (A) Ovaries from the control, (B) treated with M125, (C) treated with M250, and (d) treated with M500. The figures show primordial, primary, secondary and Graffian follicles (magnification: ×10, scale bar =100 μm). Primordial follicles (PF I), the primary follicles(PF II), the secondary follicles (SF), the Graffian follicles (GF), MOFs, multi-oocyte follicles.

Then, the numbers of the ovarian follicles in each group were compared, including PFI, PFII, SF and GF, respectively (Fig. 6). The comparison results showed that supplemental MRJPs resulted in decrease of PFI, PFII significantly, and increase of SF and griffin GF,compared to CK (P < 0.001). The SF in M125, M250 and M250 were 10.75, 12.75, 9.88, which were increased by 50.7% (P = 0.034), 78.8%(P = 0.001) and 38.6% compared to CK (7.13) (P = 0.148), respectively, The mean GF in M125 and M250 and M250 were 3.50, 4.37 and 1.25, which were increased by 600.0%, 774.0% and 150.0% compared to CK (0.50) (P < 0.001), respectively.

Fig. 6. Comparison on the number of PF I, PF II, SF and GF of different groups. Different lowercase letters (a–b) above bars indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range tests.

3.7. Effects of MRJPs on the serum hormone levels

To determine whether MRJPs supplementation affect ovarian hormones in female mice, the serum E2and P levels were assayed.As shown in Fig. 7A, the serum E2levels in M125, M250 and M500 were (25.16 ± 2.86), (28.19 ± 2.03) and (22.42 ± 1.74) pg/mL,which were significantly elevated by 47.1%, 64.9% and 31.3% (P< 0.001) compared to CK ((17.1 ± 0.78) pg/mL), respectively. As shown in Fig. 6B, the serum P levels in M125, M250 and M500 were (7.21 ± 2.68), (13.59 ± 3.82) and (6.90 ± 1.90) ng/mL, which were significantly elevated by 13.5% (P = 0.949), 114.0% (P = 0.001)and 8.7% (P = 0.986) compared to CK ((6.35 ± 2.28) ng/mL), respectively. A significant increase (P = 0.001) of serum P concentration was observed in M250 compared to CK (Fig. 7B). The results suggest that supplemental MRJPs could stimulate the secretion of ovarian steroids, and the best benefit supplemental dose of MRJPs is M 250.

Fig. 7. Effects of MRJPs on the serum hormones levels in female mice. A. Histogram of serum levels of estradiol (E2) and B. Histogram of serum levels of progesterone (P).Data are expressed as mean ± SE (n = 6). Different lowercase letters (a–d) above bars indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range tests.

3.8. Effects of supplemental MRJPs on the gene expression of hormone receptors

To investigate the action molecular mechanisms by which MRJPs promote follicular development, the changes of representative genes, ERα and ERβ were measured, respectively, and the relative expression of two genes were depicted in Fig. 8. The results showed that supplemental MRJPs had no significant effects on the expression of ERα (P = 0.583), however, the expression of ERβ in female mice fed MRJPs in all treatment was significantly increased compared to CK (P < 0.001), which indicated that supplemental MRJPs could raise hormone secretion level to promote ovary development mainly via upregulating expression of ERβ gene.

Fig. 8. Effects of MRJPs on the expression of ERα mRNA and ERβ mRNA of ovaries.A. Effect of MRJPs on the expression of ERα mRNA of ovaries. B. Effect of MRJPs on the expression of ERβ mRNA of ovaries.

3.9. Effects of MRJPs supplementation on antioxidant properties in ovary issues

The effects of MRJPs on antioxidant activities were showed in Table 2. The MDA levels of ovary issues were assessed, given that MDA is indicative of lipid peroxidation and oxidative damage. We found that the MDA level in the M125 and M250 groups were decreased by 24.6% (P = 0.023) and 26.74% (P = 0.015) compared to CK, respectively. However, the activities of SOD in the M125 and M250 were increased by 11.33% (P = 0.049) and 18.14% (P =0.002) compared to CK, respectively, which showed significant differences. Similarly, the GSH-Px activities of M125 and M250 were increased by 19.19% (P = 0.033) and 19.6% (P = 0.025), respectively,which were significantly higher than that in CK.

4. Discussion

Puberty is the final stage of maturation of the hypothalamicpituitary-gonadal axis, culminating in an adult phenotype, and is marked by increasing levels of hormone steroids [35]. Though the onset of puberty is a genetically driven event, it can be changed by environmental factors, nutritional status and metabolic status[36]. In various mammals, puberty onset could be induced by the injection of gonadotropin-releasing hormone (GnRH), follicle stimulating hormone, luteinizing hormone or analogous compounds of general excitatory neurotransmitter of the central nervous system[37]. The development of sexual maturity involves the interaction of gonadotropin releasing produced and released from the pituitary in response to GnRH and E2produced by the ovaries in response to increasing levels of gonadotropins [38]. The estrogenic and inducing effects on some physiological parameters of the onset of puberty (age and phase of the estrous cycle at vaginal opening)has been evaluated. Consumption of dietary estrogenic compounds in immature female animals promotes the sexual maturation [39].Our results showed that neonatal supplementation with MRJPs led to an early onset of vaginal opening, and dramatically acceleration of the puberty onset in immature FM, suggesting MRJPs possesses estrogenic-like effect.

The reproductive system is one of the basic systems regulated by hormones in organisms. Ovarian steroids play key roles in maturation and growth of reproductive organ. Follicular development is a critical event in mammalian female reproduction. Estrogen functions may act via two estrogen receptors, ERα and ERβ [40]. E2and P are two important hormones required for follicle development. Mishima et al. [20] found that some components in RJ can bind to estrogen receptors and stimulate estrogen-sensitive gene expression through in vitro molecular pharmacology experiments,indicating that RJ has a significant estrogenic effect. However, the specific active ingredient mediating the effect in RJ remains unclear.It seems that RJ also exert estrogenic effect in the immature FM[21]. In this study, the estrogenic action of MRJPs in FM was further illustrated. We found that oral MRJPs administration may cause remarkable increase in the serum steroid hormones including E2and P in FM, and the action of MRJPs raising hormone secretion level is mainly via modulating the recruitment of ERα, ERβ and co-activators to target genes. Moreover, the estrogen-like effect of MRJPs was associated with its supplemental dose. M250 presented the significant estrogen-like effect, while M125 and M500 showed worse effects than M250.

Reactive oxygen species (ROS) are formed continuously in cells as a consequence of both oxidative biochemical reactions and external factors. In females, changes of ROS levels are critical in reproductive functions, including oocyte maturation, ovarian steroidogenesis, follicular development, and ovulation. However,excess production of ROS could result in adverse effects on cell function and elevating risk of ovarian physiology. Accumulating data have shown that ROS contributes to the accelerated aging of oocyte and deteriorates oocyte quality [41]. The antioxidant properties of RJ have been con firmed in previous studies. It was con firmed that the cisplatin-induced changes in histopathologic findings of kidneys could be partially reversed by treatment with RJ [42]. Furthermore, once RJ is hydrolyzed using enzyme, the hydrolysate possesses much higher antioxidative activity and scavenging activity against active oxygen species [43]. Our antioxidation assay results suggested that the MRJPs extracted from RJ have excellent free radical scavenging and anti-oxidative properties by increasing the activity of SOD and GSH-Px in ovary cells, and decreasing the concentration of MDA, con firming the antioxidant potential of MRJPs in oxidative stress of female ovary issues. These results are in agreement with the finding of previous study reported that enzyme-treated RJ (ERJ) significantly increased the activity of the antioxidant enzyme SOD and the level of the GSH-Px, indicating that ERJ has high potential as an antioxidant agent using in human and animal diets [44].

Moreover, our results in ovarian histology indicated that supplemental MRJPs increased the number of SF and GF, and high dose of MRJPs induced presence of MOFs, which suggesting that MRJPs exert an estrogen-like activity on follicular growth. This activity was similar to that of isoflavone genistein from soy and soy-derived products. Alterations in ovarian differentiation including formation of MOFs were observed in mice treated neonatally with genistein[45]. Subcutaneous injection of genistein to neonatal mice induced the formation of multi-oocyte follicles and delayed the primordial follicle assembly in the ovaries [46]. Previous reports have showed that RJ possesses the activity to promote ovarian follicular development in immature rat [20], and three fatty acids including 10 HDA from RJ exerted estrogenic activity in immature rats[21]. Our current projects found that MRJPs of RJ derived products induced presence of MOFs in mice firstly, which suggested that MRJPs might be the major components of estrogen-like activity in RJ, which increased endogenous estrogen levels in animals.It was con firmed that the supplemental MRJP contained protein with more than 80% and trace 10-HDA in our analysis though the estrogenic activities of the fatty acids from RJ had been proved previously [20]. In future, the mechanisms of how MRJPs induced the estrogen-like activity and active comparison between MRJPs and the fatty acids from RJ such as 10-HDA should be investigated further.

5. Conclusions

In summary, MRJPs supplementation accelerate the onset of puberty, and promote follicular development in immature FM. The reproductive performance promotion of MRJPs was associated with raising estrogenic activity and antioxidant potential to reproductive system, upregulating expression of ERβ gene to raise hormone secretion and promote ovary development in FM. This study provides new evidence that MRJPs extracted from RJ might be a new potential supplemental proteins to promote fertility parameters,and supplemental MRJPs might be an effective approach to treat infertility in future.

Declaration of Competing Interest

The authors have declared no conflicts of interest. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.

Acknowledgements

The authors are grateful to Dr. Quanwei Wei from Nanjing Agricultural University, Nanjing, China for his technical assistance. This work was supported by the National Natural Science Foundation of China (no. 31271848)