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Declined expressing mRNA of beta-defensin 108 from epididymis is associated with decreased sperm motility in blue fox (Vulpes lagopus)

Abstract

Background

Fecundity is important for farm blue fox (Vulpes lagopus), who with asthenospermia have be a problem in some of farms in China. A key symptom of asthenospermia is decreased sperm motility. The decreased secreting beta-defensin108 (vBD108) of blue fox is speculated be related to asthenospermia. To clarify this idea, the mRNA expression of vBD108 in testis and epididymis of blue foxes with asthenospermia were detected and compared to the healthy one. The antibody was prepared and analyzed by immunohistochemistry.

Results

The vBD108 in testis and epididymis was found both in blue fox with asthenospermia and healthy group by the method of immunohistochemistry. The expression of vBD108 mRNA in testes (Pā€‰<ā€‰0.05) and epididymal corpus (Pā€‰<ā€‰0.0001) in asthenospermia group was lower than that in healthy group.

Conclusions

These results suggested that vBD108 deficiency may related to blue fox asthenospermia. Meanwhile, the study on the blue fox vBD108 provides a hopeful direction to explore the pathogenesis of blue fox asthenospermia in the future.

Background

Ī²-defensins, well known cationic antimicrobial peptide rich in cysteine [1,2,3,4], belonging to a family of host-defense peptides, produced by multiple epithelial tissues and immune cells, played an important role in the innate immune response [5, 6]. Although most of the studies on Ī²-defensins focused on their antibacterial and anti-tumor activities, there is growing evidence that Ī²-defensins play a special role on motility of sperm in the epididymal of mammals [7, 8].

The addition of cauda epididymal fluid (CEF) of cattle containing Ī²-defensin 126 (BBD126) can significantly improve the motility of bovine spermatozoa [9]. The results of some studies suggested that human Ī²-defensin 1 (hBD-1) can affect the quality of sperm and improve sperm motility when exogenously added [10, 11]. Inhibition of the expression of Bin1b, a rat epididymis-specific Ī²-defensin with antimicrobial activity [12], resulted in a decrease in the binding of Bin1b to sperm and a significant decrease in sperm motility and progressive movement [8]. The epididymal specific Ī²-defensin 15 (Defb15) was down regulated in vivo, which would result significantly decreased the total motility and progressive motility of rat spermatozoa [7].

Some farm blue fox with low sperm motility was paid attention and it always be called asthenospermia [10]. As the Ī²-defensin is involved in the acquisition of sperm motility, the asthenospermia of blue fox may be related to the decrease of Ī²-defensin secretion was suspected. The object of this study is to confirm if the Ī²-defensin 108, especially in testis and epididymis, is related to the sperm motility.

Results

Detection of vBD108 protein in testis and epididymis

These animals did not use other drugs or other experiments before the experiment. In the immunohistochemical experiment, three healthy blue foxes (3/5) and three blue foxes with asthenospermia (3/5) were used. The reason is that about half of the samples were randomly selected for immunohistochemical detection. The results of immunohistochemical detection of vBD108 were shown in Fig. 1. Staining of the testis and caput, corpus and cauda of the epididymis from the control group without primary antibody was negative. Brown coloration indicative of positive staining was found both in the testis, caput epididymis, corpus epididymis as well as in the cauda epididymis of the asthenospermia blue fox and healthy one compare to the control group.

Fig. 1
figure 1

Immunohistochemical analysis of vBD108 expression in testis and epididymis. Tissue sections from both healthy and asthenospermia blue fox were stained with anti-vBD108 antibody except control group. Bar=17.06 Āµm. Representative images of tissue sections from n=3 blue foxes were shown. a: Testis; b: Caput; c: Corpus; d: Cauda

Detection of mRNA expression level of vBD108 in testis and epididymis of asthenospermia and the healthy blue fox

The results of immunohistochemistry showed that there was a difference in the expression of vBD108 between healthy blue fox and asthenospermia blue fox. We used qPCR technique to quantitatively analyze the expression levels of vBD108 in testes, and caput, corpus and cauda of epididymis of asthenospermia blue fox and the healthy one. The expression level of vBD108 mRNA in testes and epididymis of blue fox was analyzed as shown in Fig. 2. We found that the relative expression of vBD108 both in testes (Pā€‰<ā€‰0.05) and corpus epididymis (Pā€‰<ā€‰0.0001) of healthy blue fox was significantly higher than that of blue fox with asthenospermia. Among the healthy fox and asthenospertic one, the difference of vBD108 in corpus of epididymis is especially apparently (Pā€‰<ā€‰0.0001).

Fig. 2
figure 2

Relative expression of vBD108 in testis and epididymis of asthenospermia and healthy blue fox. Data are expressed as the mean Ā± SD, n=3. The difference between averages is determined by using Sidakā€™s multiple comparisons test (*, Pā€‰<ā€‰0.05; ****, Pā€‰<ā€‰0.0001)

Discussion

The epididymal lumen environment is an androgen response organ composed of specific ions, small organic molecules and proteins responsible for sperm maturation and storage [13]. In addition to Ī²-defensin, androgen also plays an important role in several physiological processes of epididymis. Several studies have reported that androgens may regulate some Ī²-defensins [14]. Two mouse Ī²-defensins (Defb41 and Defb42) are regulated by androgens by Q-RT-PCR and in situ hybridization [15]. Sperm-associated antigen 11A is expressed exclusively in the principal cells of the mouse caput epididymis in an androgen-dependent manner [16]. Human epididymis specifically expresses Ī²-defensin 118 (DEFB118) (formerly ESC42) is regulated by androgen [17]. Six genes (Defb18, 19, 20, 39, 41 and 42) of mouse epididymal Ī²-defensin were identified by bilateral orchiectomy and androgen supplementation, which were completely regulated by androgen [18]. Moreover, the time difference of seasonal expression is also the characteristic of Ī²-defensin. The mRNA level of SPAG11A in the epididymis of wild ground squirrels in breeding season was significantly higher than that in non-breeding season [16]. A lot of studies have shown that the expression of Ī²-defensin in testes and epididymis of different species (rat, mouse, sheep, goat) is time-dependent. The expression of sheep Defb124 gene in sheep testes and epididymis and the goat beta-defensins 104a in the epididymis have age differences [19, 20]. Likewise, the expression of Ī²-defensins (Defb15 and āˆ’ā€‰49) in rat epididymis and Defb41 in mouse testes are different during postnatal development [7, 15, 21]. To sum up, we speculate that vBD108 may have the same differences in age distribution and androgen dependence and seasonal expression as these Ī²-defensins. However, these functions of vBD108 still need to be tested by experiments in the future.

A great number of studies have shown that Ī²-defensin is involved in antimicrobial activity and regulating immune host [5, 22]. Loss of Ī²-defensin gene expression can lead to asthenospermia, which is usually associated with low sperm motility [10, 23]. Sperm motility is considered to be one of the most important sperm functions affecting natural conception [24]. More and more studies have shown that mammalian Ī²-defensin has an effect on sperm function. It is worth noting that after sperm are produced from the testis, they gain their motility and fertilization ability through the epididymis, which is called sperm maturation [13, 25]. Biology generally divides the epididymis into caput, corpus and cauda [9, 26, 27], which created unique and dynamic physiological environment provides conditions for sperm maturation [28,29,30,31]. A variety of region-specific epididymal Ī²-defensins contribute to different microenvironments of epididymal sperm maturation in different segments were found [6]. The expression of vBD108 protein in epididymis of blue fox with asthenospermia have an apparently difference compare with the healthy one, it suggests that vBD108, as a molecule of epididymal microenvironment, may be involved in the acquisition of sperm motility. This view is supported by some reports. A rat epididymal specific Ī²-defensin (Bin1b) [12] can bind to sperm heads in different regions of the epididymis in different binding modes has been reported. Moreover, Bin1b is considered to be related to epididymal sperm maturation [8]. Human Ī²-defensin 1 (DEFB1) interacts with chemokine receptor type 6 (CCR6) in spermatozoa, triggering Ca2+ mobilization, which is key for sperm motility [10]. Ī²ā€‘defensin DEFB126, a multifunctional glycoprotein, is adsorbed to the sperm surface during epididymal maturation [32]. All in all, these findings suggest that members of vBD108 may be involved not only in antibacterial and host immunity, but also in the regulation of sperm motility.

Although our experimental results found that vBD108 may be related to azoospermia in blue foxes, we need more experiments to determine the role of vBD108 in sperm motility and male reproduction. We plan to clarify this problem through several aspects in following works. Firstly, vBD108 antibody or vBD108 protein are co-cultured with sperm to observe the changes of sperm motility. Secondly, the expression of vBD108 gene in the epididymis of blue fox was knocked down by RNAi technique, and the changes of the binding ability of vBD108 to sperm and the binding ability between sperm and ovum were analyzed. Finally, mating experiments were conducted to evaluate the changes in fertility of male blue fox with down-regulated vBD108.

Conclusions

The results of this study demonstrate that low vBD108 in corpus of epididymis was associated to asthenospermia and might be causal linked to low sperm motility. Meanwhile, the study on the blue fox vBD108 provides a hopeful direction to explore the pathogenesis of blue fox asthenospermia in the future.

Methods

Animals and tissue collection

The five healthy and eight asthenospermia blue foxes are provided by Harbin Hualong Blue Fox breeding Co., Ltd. The age of the animals were two years old. The weight of the animals was from 17 to 22 kg. These experimental animals were all male. Wu et al. used sperm motility analysis system (IVOS) to detect sperm motility in fresh semen. Sperm forward motility is less than 50% or fast straight forward movement is less than 25%, which is called low sperm motility, also known as asthenospermia [33]. According to the International Animal Welfare Law, the blue fox was euthanized by intravenous injection of excessive pentobarbital sodium. The testes, and caput, corpus and cauda of epididymes were collected in April 2017 in College of Wildlife and Protected Area, Northeast Forestry University, Harbin, China. The samples were soaked in RNA preservation solution and stored at -80 Ā°C. In addition, the tissue samples of each sample were cut into tissue blocks of 0.5 cmā€‰Ć—ā€‰0.5 cmā€‰Ć—ā€‰0.2 cm and fixed in 4% neutral formaldehyde fixation solution. Four SPF New Zealand rabbits were purchased from the Harbin Veterinary Research Institute, CAAS. The age of the rabbits ranged from 1 to 1.5 years and the weight of the animals were from 1.45 to 1.55 kg. Rabbits were fed with rabbit feed (SPF experimental rabbit feed) in 37 Ā°C laboratory animal room. Two rabbits were kept in each cage and they had free access to water. The rabbits were euthanized after excessive intravenous injection of pentobarbital sodium at the end of the experiment.

Preparation of polyclonal antibody against rabbit vBD108

A custom monoclonal antibody specific for vBD108 was commercially generated by Genscript using a standard protocol. The synthesized vBD108 protein was diluted with sterilized PBS (Takara, Dalian) buffer and emulsified with Freund Adjuvant (Takara, Dalian) at a volume ratio of 1:1, and then used for inoculation. 1 ml of immune antigen emulsion (0.25 mg /ml) in 200ā€“300 Āµl per each site was injected into the abdominal cavity of rabbits. Operate in the same way every 14 to 16 days. Rabbits were immunized three times, the first one was immunized with Freund complete adjuvant, and the last two times, Freund incomplete adjuvant was selected [34]. After the third immunization, the blood was collected from the auricular vein on the 12th day and the serum was separated.

Enzyme-linked immunosorbent assay

The titer of serum was determined by indirect ELISA [35] with respective pre-immunized serum as negative control. In short, the gradient diluted vBD108 was used to coat the enzyme labeling plate transversely, and the gradient diluted rabbit vBD108 positive serum and negative serum were added longitudinally to the enzyme labeling plate. The absorbance value was measured and the optional coating concentration of vBD108 and the best working concentration of serum were determined. Then the antibody titer of vBD108 was determined by the same method with the determined optimal antigen coating concentration. The ratio of OD490nm in positive serum to OD490nm in negative serum was between 1.5 and 3.0, which was the zero point of positive reaction, and the corresponding serum dilution was the antibody titer of serum.

Immunohistochemical detection of vBD108 in testis and epididymis of blue fox with asthenospermia and the healthy one

The samples of blue foxes with asthenospermia (n=3) and the healthy one (n=3) were used by immunohistochemistry. The fixed tissue samples were dehydrated with different concentrations of ethanol, the dehydrated tissues were treated with anhydrous ethanol and xylene 1:1 mixture for 30 min, and xylene solution 20 min for twice. After transparent operation, the tissue needs to be soaked in wax for 3 hours and embedded 3ā€“5 Āµm thick slices, fixed on chromic acid-treated slides, spread them in a pool at 30 Ā°C, bake at 37 Ā°C overnight or 60 Ā°C for 2 hours. After tissue dewaxing, the slides were heated in sodium citrate at 98 Ā°C in 10 min for antigen repair, 3% H2O2 for 10 min, 5 min washing for 3 times, 1ā€‰Ć—ā€‰PBS for 5 minutes. Goat serum was diluted 10 times with 1ā€‰Ć—ā€‰PBS and sealed for one hour. The primary antibody was diluted according to 1:1000, each piece was 100 Āµl (wax circle), overnight at 4 Ā°C (recovery of the primary antibody), and 5 min washed with 1ā€‰Ć—ā€‰PBS for three times after incubation.

The slides were incubated with the second antibody (HRP labeled goat anti-rabbit IgG) 37 Ā°C for 1 hour with the diluent in the DAB chromogenic kit (Takara, Dalian). H2O2 was added to the diluted DAB chromogenic solution according to the ratio of 1:1000, and then added to the glass slide for color development. Re-dye with hematoxylin for one minute, add dilute hydrochloric acid to return to red, and then return to blue in light ammonia water. Finally, it was dehydrated, sealed with neutral gum and observed under microscope. Controls were subjected to the same procedure except without adding primary antibody.

Acquisition of organizational cDNA

According to the operation instructions of Trizol Reagent kit (BioTeke, Beijing), the total mRNA, nucleic acid and protein analyzer NanoDrop-2000 (Beijing) was used to determine the concentration and purity of total RNA, and then the adjusted concentration was about 0.5 Āµg/ml. According to canine Ī²-defensin 108 gene (accession: XM_003432123) in NCBI database, degenerate primer was designed using primer 5.0. The coding sequence of vBD108 was amplified by the following primers: forward primer F: 5ā€™- GAAGCCKTGTCTGCCTCTG āˆ’ā€‰3ā€™ and reverse primer R: 5ā€™- GATCATTCCTTGGGTGTAG āˆ’ā€‰3ā€™. Reverse transcription was performed according to the instructions of HiScripĀ® II Q RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme, Nanjing). The first step was as follows: 20 Āµl total reaction system, including 4Ɨ gDNA wiper Mix 4 Āµl, 12 Āµl total RNA extracted from each tissue, 42 Ā°C for 2 min. The second step: 5Ɨ HiScript II qRT SuperMix II 4Āµ l, the first step reaction solution 16 Āµl, reverse transcription conditions: 50 Ā°C for 15 min, 85 Ā°C for 5 sec, products were stored at -20 Ā°C.

qPCR

In order to detect the expression of vBD108 in testis and epididymis of blue fox, the primers for qPCR detection of fox vBD108 gene (F: 5ā€™-CTCGCCCTGCTCTTCTTTCT-3ā€™, R: 5ā€™-CGTGGCCATTTAAACACCTC āˆ’ā€‰3ā€™) were designed according to canine Ī²-defensin 108 gene (accession: XM_003432123) in NCBI database. According to the reaction system and reaction conditions of AceQ qPCR SYBR Green Master Mix kit (Vazyme, Nanjing), real-time quantitative PCR reaction was carried out. The cDNA obtained in the experiment was used as a template, and the qPCR detection primers mentioned above were added to the 96-well plate (with repetitive holes) dedicated for fluorescent quantitative PCR, and the following reagents were added at one time. The upstream and downstream primers were respectively 1 Āµl, SYBR Green Master Mix (2Ɨ) 7.5 Āµl, cDNA solution 3 Āµl, ddH2O supplement to the final reaction volume of 16 Āµl. GAPDH was used as the internal reference, the sequence of it was amplified by the following primers: the forward primer F: 5ā€™- AACATCATCCCTGCTTCCAC āˆ’ā€‰3ā€™ and the reverse primer R: 5ā€™- ATGCCTGCTTCACTACCTTCTT āˆ’ā€‰3ā€™. The fluorescence quantitative results were all processed by 2āˆ’ā–³ā–³CT method, and the relative expression of target gene was analyzed by GraphPad Prism Version 8.0.2.

Statistical analysis

We performed statistical analysis using GraphPad Prism Version 8.0.2. Results are shown as mean Ā± standard deviation. Data correlation analysis was performed between groups. We considered results to be significant at Pā€‰<ā€‰0.05.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the GenBank (https://www.ncbi.nlm.nih.gov/nuccore/XM_003432123) and the accession number is XM_003432123.

Abbreviations

mRNA:

Messenger ribonucleic acid.

CEF:

Cauda epididymal fluid

qPCR:

Real-timeĀ Quantitative PCR Detecting System

CCR6:

Chemokine receptor type 6

RNA:

Ribonucleic acid

SPF:

Specific pathogen free

CAAS:

Chinese Academy of Agricultural Sciences

H2O2 :

Hydrogen peroxide

PBS:

Phosphate buffered solution

ELISA:

Enzyme-linked immunosorbent assay

cDNA:

Complementary deoxyribonucleic acid

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

SD:

Standard deviation

References

  1. Aram R, Chan PTK, Cyr DG. Beta-defensin126 is correlated with sperm motility in fertile and infertile men. Biol Reprod. 2019;102(1):92ā€“101.

    Google ScholarĀ 

  2. Dorin JR, Barratt CL. Importance of Ī²-defensins in sperm function. Mol Hum Reprod. 2014;20(9):821ā€“26.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  3. Ribeiro CM, Silva EJ, Hinton BT, Avellar MC. Ī²-defensins and the epididymis: contrasting influences of prenatal, postnatal, and adult scenarios. Asian J Androl. 2016;18(2):323ā€“28.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  4. White SH, Wimley WC, Selsted ME. Structure, function, and membrane integration of defensins. Curr Opin in Struct Biol. 1995;5(4):521ā€“27.

    ArticleĀ  CASĀ  Google ScholarĀ 

  5. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol. 2003;3(9):710ā€“20.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  6. Yamaguchi Y, Nagase T, Makita R, Fukuhara S, Tomita T, Tominaga T, Kurihara H, Ouchi Y. Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice. J Immunol. 2002;169(5):2516ā€“23.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  7. Zhao Y, Diao H, Ni Z, Hu S, Yu H, Zhang Y. The epididymis-specific antimicrobial peptide Ī²-defensin 15 is required for sperm motility and male fertility in the rat (Rattus norvegicus). Cell Mol Life Sci. 2011;68(4):697ā€“708.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  8. Zhou CX, Zhang YL, Xiao L, Zheng M, Leung KM, Chan MY, Lo PS, Tsang LL, Wong HY, Ho LS, Chung YW, Chan HC. An epididymis-specific Ī²-defensin is important for the initiation of sperm maturation. Nat Cell Biol. 2004;6(5):458ā€“64.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  9. Fernandez-fuertes B, Narciandi F, Ofarrelly C, Kelly AK, Fair S, Meade KG, Lonergan P. Cauda Epididymis-Specific Beta-Defensin 126 Promotes Sperm Motility but Not Fertilizing Ability in Cattle. Biol Reprod. 2016;95(6):122.

    ArticleĀ  PubMedĀ  PubMed CentralĀ  CASĀ  Google ScholarĀ 

  10. Diao R, Fok KL, Chen H, Yu MK, Duan Y, Chung CM, Li Z, Wu H, Li Z, Zhang H, Ji Z, Zhen W, Ng CF, Gui Y, Cai Z, Chan HC. Deficient human Ī²-defensin 1 underlies male infertility associated with poor sperm motility and genital tract infection. Sci Transl Med. 2014;6(249):249ra108.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  11. Zupin L, Polesello V, Martinelli M, Luppi S, Giolo E, Zito G, Romano F, Segat L, Crovella S, Ricci G. Human Ī²-defensin 1 in follicular fluid and semen: impact on fertility. J Assist Reprod Genet. 2019;36(4):787ā€“97.

    ArticleĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  12. Li P, Chan HC, He B, So SC, Chung YW, Shang Q, Zhang YD, Zhang YL. An antimicrobial peptide gene found in the male reproductive system of rats. Science. 2001;291(5509):1783ā€“85.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  13. Johnston DS, Jelinsky SA, Bang HJ, DiCandeloro P, Wilson E, Kopf GS, Turner TT. The mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis. Biol Reprod. 2005;73(3):404ā€“13.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  14. Oh J, Lee J, Woo JM, Choi E, Park I, Han C, Baek N, Lee H, Kim DH, Cho C. Systematic identification and integrative analysis of novel genes expressed specifically or predominantly in mouse epididymis. BMC Genom. 2006;7:314.

    ArticleĀ  CASĀ  Google ScholarĀ 

  15. Jalkanen J, Huhtaniemi I, Poutanen M. Discovery and characterization of new epididymis-specific beta-defensins in mice. Biochim Biophys Acta. 2005;1730(1):22ā€“30.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  16. Yu W, Zhang Z, Liu P, Yang X, Zhang H, Yuan Z, Han Y, Weng Q. Seasonal expressions of SPAG11A and androgen receptor in the epididymis of the wild ground squirrels (Citellus dauricus Brandt). Eur J Histochem. 2020;64(2):3111.

    ArticleĀ  CASĀ  PubMed CentralĀ  Google ScholarĀ 

  17. Yenugu S, Hamil KG, Radhakrishnan Y, French FS, Hall SH. The androgen-regulated epididymal sperm-binding protein, human beta-defensin 118 (DEFB118) (formerly ESC42), is an antimicrobial beta-defensin. Endocrinology. 2004;145(7):3165ā€“73.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  18. Hu SG, Zou M, Yao GX, Ma WB, Zhu QL, Li XQ, Chen ZJ, Sun Y. Androgenic regulation of beta-defensins in the mouse epididymis. Reprod Biol Endocrinol. 2014;12:76.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  19. Li Z, Zhang GL, Guo LN, Zhang CX. Expression of Defb124 Gene in Testis, Epididymidis at Different Stages of Sheep. China Herbivore Science. 2014;34(04):5ā€“8. (in Chinese).

    Google ScholarĀ 

  20. Guo LN, Zhang GL, Ren YS, Jiang YS, Liu WZ, Zhang CX: Localization and expression character of Beta-Defensins 104a in different days of male goat testis and epididymis. J SHANXI AGRIC, UNIV(Natural Science Edition). 2017;37(12):866 ā€“ 71. (in Chinese).

  21. Patil AA, Cai Y, Sang Y, Blecha F, Zhang G. Cross-species analysis of the mammalian beta-defensin gene family: presence of syntenic gene clusters and preferential expression in the male reproductive tract. Physiol Genomics. 2005;23(1):5ā€“17.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  22. Hall SH, Hamil KG, French FS. Host defense proteins of the male reproductive tract. J Androl. 2002;23(5):585ā€“97.

    CASĀ  PubMedĀ  Google ScholarĀ 

  23. Curi SM, Ariagno JI, Chenlo PH, Mendeluk GR, Pugliese MN, Sardi Segovia LM, Repetto HE, Blanco AM. Asthenozoospermia:analysis of a large population. Arch of Androl. 2003;49(5):343ā€“49.

    ArticleĀ  CASĀ  Google ScholarĀ 

  24. Hunault CC, Habbema JD, Eijkemans MJ, Collins JA, Evers JL, te Velde ER. Two new prediction rules for spontaneous pregnancy leading to live birth among subfertile couples, based on the synthesis of three previous models. Hum Reprod. 2004;19(9):2019ā€“26.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  25. Cooper TG. Sperm maturation in the epididymis: a new look at an old problem. Asian J Androl. 2007;9(4):533ā€“9.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  26. Chauvin TR, Griswold MD. Androgen-regulated genes in the murine epididymis. Biol Reprod. 2004;71(2):560ā€“69.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  27. Jelinsky SA, Turner TT, Bang HJ, Finger JN, Solarz MK, Wilson E, Brown EL, Kopf GS, Johnston DS. The rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides. Biol Reprod. 2007;76(4):561ā€“70.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  28. Breton S, Nair AV, Battistone MA. Epithelial dynamics in the epididymis: role in the maturation, protection, and storage of spermatozoa. Andrology. 2019;7(5):631ā€“43.

    CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  29. Cornwall GA. Role of posttranslational protein modifications in epididymal sperm maturation and extracellular quality control. Adv Exp Med Biol. 2014;759:159ā€“80.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  30. Hinton BT, Palladino MA. Epididymal epithelium: Its contribution to the formation of a luminal fluid microenvironment. Microsc Res Tech. 1995;30(1):67ā€“81.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  31. Jervis KM, Robaire B. Dynamic changes in gene expression along the rat epididymis. Biol Reprod. 2001;65(3):696ā€“703.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  32. Tollner TL, Bevins CL, Cherr GN. Multifunctional glycoprotein DEFB126ā€”a curious story of defensin-clad spermatozoa. Nat Rev Urol. 2012;9(7):365ā€“75.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  33. Wu RZ, Hao XM, Liu ZP, Han HS. Sperm motility and ultrastructural changes in blue fox semen before and after freezing. Heilongjiang Animal Science and Veterinary Medicine. 2019;(23):131ā€“134 + 139. (in Chinese).

  34. Hancock DC, Oā€™Reilly NJ: Production of polyclonal antibodies in rabbits. In: Burns R, editor. Immunochemical Protocols. Methods Mol Biol; 2005. p.Ā 27ā€“40.

  35. Crowther JR. The ELISA guidebook. Methods Mol Biol; 2000.

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Acknowledgements

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Funding

This research was supported by the National Key Research and Development Program of China (No.2018YFD0502201) and the Fundamental Research Funds for the Central Universities (NO.2572014CA02). This funding body provided monetary support only and did not have any input into the design of the study, collection, analysis, and interpretation of data.

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Contributions

LT and ZH contributed to conception and design of the study. ZL, TL is responsible for sample collection. TL participated in the experimental procedures including immunohistochemistry. WP and LL are responsible for data analysis and interpretation and manuscript writing and revisions. All authors have read and approved the final product. As joint first authors, both PW and TL contributed equally to the research work.

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Correspondence to Li-hong Tian or Zhi-jun Hou.

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All experimental designs and animals handling were approved by the Laboratory Animal Management and Ethics Committee of Northeast Forestry University (2020023).

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Wu, P., Liu, Tl., Li, Ll. et al. Declined expressing mRNA of beta-defensin 108 from epididymis is associated with decreased sperm motility in blue fox (Vulpes lagopus). BMC Vet Res 17, 12 (2021). https://doi.org/10.1186/s12917-020-02697-6

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