Original Research Article

Lactoferrin expression and secretion in the stallionepididymis

Christopher A. Pearl a,b,*, Janet F. Roser b

aDepartment of Biological Sciences, Western Michigan University, 1903 West Michigan Avenue, Kalamazoo,MI 49008, USAbDepartment of Animal Science, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA

r e p r o d u c t i v e b i o l o g y 1 4 ( 2 0 1 4 ) 1 4 8 – 1 5 4

a r t i c l e i n f o

Article history:

Received 5 June 2013

Accepted 15 October 2013

Available online 1 November 2013

Keywords:

Lactoferrin

Epididymis

Stallion

Estrogen

Sperm

a b s t r a c t

Lactoferrin is one of the most abundant proteins secreted by the stallion epididymis, but its

cellular localization and regulation remain unknown. This study was designed to address

the following objectives: (1) identify the epididymal cell types producing lactoferrin in pre-

pubertal, peri-pubertal and post-pubertal animals; (2) demonstrate that lactoferrin binds to

stallion sperm; and (3) determine if testosterone and estradiol regulate lactoferrin secretion

in vitro. Using an immunohistochemical method, lactoferrin was localized in the cytoplasm

of principal cells in the corpus and cauda of peri- and post-pubertal animals. The epididymis

of pre-pubertal animals did not express lactoferrin. Immunolabeling of lactoferrin was also

observed on the mid-piece and tail of the sperm. The role of estradiol and testosterone in

regulating secretion of lactoferrin in the post-pubertal epididymis was investigated using

tissue culture methods. Lactoferrin concentration in the culture media was determined by

validated enzyme-linked immunosorbent assays (ELISA). Testosterone did not increase the

concentration of lactoferrin in the media in any epididymal region. In contrast, estradiol-17b

significantly increased the concentration of lactoferrin in the media containing tissue from the

cauda. In conclusion, the expression of lactoferrin was found in the cytoplasm of principal cells

in the corpus and cauda of the epididymis in peri- and post-pubertal stallions but not pre-

pubertal stallions. Furthermore, lactoferrin binds to sperm, suggesting a biological role for

protection or regulation of sperm in the corpus and cauda. In addition, estrogen appears to

regulate lactoferrin secretion in the cauda of the epididymis in post-pubertal stallions.

# 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and

Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban &

Partner Sp. z o.o. All rights reserved.

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: http://www.elsevier.com/locate/repbio

1. Introduction

As sperm traverse the epididymis, a process that takesapproximately 10 days in mammals, they undergo maturation

* Corresponding author at: Department of Biological Sciences, Western49008, USA. Tel.: +1 269 387 5608; fax: +1 269 387 5609.

E-mail address: christopher.pearl@wmich.edu (C.A. Pearl).1642-431X/$ – see front matter # 2014 Society for Biology of ReproducPolish Academy of Sciences in Olsztyn. Published by Elsevier Urban &http://dx.doi.org/10.1016/j.repbio.2013.10.005

that includes the development of motility and membraneremodeling [1,2]. Sperm are modified as a result of sequentialpassage through the microenvironments of the efferent ducts,caput and corpus until they reach the cauda where they arestored until ejaculation [3,4]. The exact site within the

Michigan University, 1903 West Michigan Avenue, Kalamazoo, MI

tion & the Institute of Animal Reproduction and Food Research of Partner Sp. z o.o. All rights reserved.

r e p r o d u c t i v e b i o l o g y 1 4 ( 2 0 1 4 ) 1 4 8 – 1 5 4 149

epididymis where sperm become fully mature varies betweenspecies [5]. In the boar for example, approximately 45% ofsperm in the caput and corpus are morphologically maturecompared with 85% in the cauda [6]. Binding sites for theoocyte are found on most sperm in the corpus and cauda, buton only 6% of sperm from the caput [7]. The extremely lowfertilization rates obtained with sperm from the caput [8]increase from 10% to 50% and 80% to 100% with sperm from thecorpus and cauda, respectively [8,9]. In the stallion, motility ofcauda sperm was similar to ejaculated sperm and sperm wereinfertile until they entered the cauda epididymidis [10]. In therabbit, immature sperm cultured with epididymal epithelialcells showed a marked increase in fertilizing ability comparedto sperm that were not cultured with epididymal cells. Whenthese immature sperm were cultured with epididymal cellsand RNA synthesis inhibitors, sperm were unable to matureand gain fertilizing ability [11]. Results from these experimentsdemonstrated that without protein synthesis and secretion bythe epididymis, sperm were unable to complete maturationand were unable to fertilize the oocyte. The profile of secretedepididymal proteins in multiple species, including the stallion,have recently been identified and includes lactoferrin [12–14].From these studies three key observations have been made: (1)protein secretion within the epididymis shows a highlyregionalized pattern; (2) regional protein secretion is likelyintegral to proper sperm maturation within the micro-environments of the epididymal lumen; and (3) lactoferrinappears to be a key component of epididymal secretion.However, much remains unresolved in regards to regulation ofepididymal protein secretion, particularly lactoferrin.

Lactoferrin has been postulated to act as an innate immunesystem defense protein of the male reproductive tract [15] withantibacterial, antiviral, antifungal and antioxidant activity, aswell as immuno-regulatory effects [16,17]. It is also known tobe a major sperm-coating antigen of other species [18,19]. Ofparticular interest to the stallion is the fact that lactoferrin isone of the most abundant proteins secreted by the stallionepididymis representing approximately 41% of the totalprotein secretion [14] and is present in seminal plasmaranging from 42 to 453 mg/mL [20]. While lactoferrin secretionfrom the post-pubertal stallion epididymis has been demon-strated [14], the exact cell type(s) responsible for its secretionremain unclear. The presence of lactoferrin in the equineepididymis of pre-pubertal and peri-pubertal animals is alsounknown. While lactoferrin has been measured in equineseminal plasma, whether it is bound to stallion sperm has notbeen demonstrated.

It is well known that androgens influence epididymalprotein secretion [21] and it is likely that estrogens are alsoinvolved [22]. The role of estrogens in regulating epididymalprotein secretion is of particular interest in the stallion giventhe high ratio of estrogens to androgens produced by the testes[23]. Additionally, estrogens and estrogen receptors are foundthroughout the stallion epididymis [24] suggesting that proteinsecretion in the stallion epididymis may be regulated byestrogens in addition to androgens. Lactoferrin has beenshown to be up regulated by estrogens in the femalereproductive tract of humans and rodents [25–27] and onlypartially androgen regulated in the boar epididymis [28]. Theimmature mouse epididymis could be stimulated to produce

lactoferrin by treatment with estradiol [29]. However, thesteroid hormone regulation of lactoferrin in the equinereproductive tract remains unknown.

Therefore, this study was designed to address the followingobjectives: (1) identify the epididymal cell types producinglactoferrin in pre-pubertal, peri-pubertal and post-pubertalanimals; (2) demonstrate that lactoferrin binds to stallionsperm; and (3) determine if testosterone and estradiol regulatelactoferrin secretion in vitro.

2. Materials and methods

2.1. Animals

Epididymides were collected from routine castrations per-formed at the UC Davis Veterinary Medicine Teaching Hospitalor the UC Davis Department of Animal Science Horse Barn.Animal use protocols for routine castration at each site wereapproved by the Institutional Animal Care and Use Committeein accordance with American Association for the Accreditationof Laboratory Animal Care (AAALAC) at UC Davis. All animalswere of light horse breeds of various ages. Epididymides weredivided into caput, corpus and cauda and fixed in 4%paraformaldehyde (w/v) for immunohistochemistry (IHC) orprocessed for tissue culture. Epididymal regions were identi-fied based on previous reports [10,14,24]. For IHC, three pre-pubertal (5–10 months), three peri-pubertal (12–18 months)and six post-pubertal (2–8 years) animals were used; agegroupings were similar to those previously documented [24].Fixed mammary gland tissue from a lactating mare, collectedat necropsy, was provided by the Veterinary MedicineTeaching Hospital at UC Davis. Epididymal tissue from fivepost-pubertal animals was processed for tissue culture. Spermfrom the cauda of three post-pubertal stallions (2–5 years) wasrecovered by retrograde flush, washed and fixed in 4%paraformaldehyde. After fixation, aliquots of sperm weresmeared onto poly-L-lysine coated slides (VWR; Radnon, PA,USA) and stored at �20 8C for immunolocalization.

2.2. Immunohistochemical method

2.2.1. Epididymal and mammary gland tissueImmunohistochemistry for lactoferrin was performed aspreviously validated and described [30]. Briefly, fixed tissuewas paraffin embedded and sectioned at a thickness of 5 mm.Tissues were incubated overnight at 4 8C with goat anti-humanlactoferrin diluted in phosphate buffered saline (1:250; sc-14434;Santa Cruz Biotechnology, Santa Cruz, CA, USA). Followingprimary antibody incubation, sections were incubated withrabbit anti-goat biotinylated secondary antibody followed by anavidin–biotin horseradish peroxidase complex (Vector Labora-tories, Burlingame, CA, USA) for 30 min at room temperature(RT). Immunolabeling was visualized using NovaRed chroma-gen (Vector Laboratories), Immunomaster Hematoxylin coun-terstain (American Mastertech, Lodi, CA, USA) at RT andevaluated by bright field light microscopy. Sections of eachregion incubated with goat IgG (sc-2028; Santa Cruz Biotechnol-ogy), at a concentration equal to primary antibody, were used asnegative controls. Images were captured using a QImaging

Fig. 1 – Immunolabeling of lactoferrin in the equinemammary gland. Lactoferrin was expressed and secretedby the alveolar cells (arrows) in the lactating mammarygland (A). No immunolabeling was observed in negativecontrol sections (B). Scale bar: 50 mm.

r e p r o d u c t i v e b i o l o g y 1 4 ( 2 0 1 4 ) 1 4 8 – 1 5 4150

Micropublisher 3.3 digital camera and QCapture Pro software(QImaging Corporation, Burnaby, BC, Canada). Intensity ofimmunolabeling was scored as negative (�), weak positive (+),positive (++) or strong positive (+++).

2.2.2. SpermSperm slides were rehydrated with deionized water andincubated for 10 min in 200 mM glycine in water. After aphosphate buffered saline wash, slides were processed forimmunolocalization of lactoferrin following the same proceduredescribedfor epididymal tissue exceptthat the primary antibodywas used at 1:50 and the incubation was 3 h at RT. Spermincubated with goat IgG instead of primary antibody were usedas negative controls. Images were captured using a QImagingMicropublisher 3.3 digital camera and QCapture Pro software.

2.3. Epididymal tissue culture

Immediately following castration, epididymides from fivepost-pubertal stallions were processed for tissue culture.Epididymides were processed in Hanks buffered salinesolution (HBSS) under sterile conditions. All fat was removedand the epididymides were divided into the caput, corpus, andcauda. The tubules were peeled away from the capsule,uncoiled and the interstitial connective tissue removed.Approximately 50 mg pieces of uncoiled tubules were mincedinto fragments of 1 mm3 or smaller and washed in Dulbecco'sModified Eagle Medium (DMEM). Tissue culture media wasDMEM (Sigma, St. Louis, MO, USA) supplemented with 2 mMcarnitine (Sigma) and 20 mM HEPES (Sigma). Tissue wascultured for 6 h in microcentrifuge tubes with gas permeablemembranes at 32 8C under 95% O2, 5% CO2 in mediasupplemented with 0.1, 1.0 or 10 mM testosterone (Steraloids,Newport, RI, USA) or 0.1, 1.0, 10 mM estradiol 17-b (Steraloids).The minimum time required for increased protein synthesisand secretion via the classical genomic steroid receptorpathway is 30–60 min [31]; thus newly synthesized lactoferrincould be present in the culture medium approximately 1 hafter culture began. Additionally, a previous report using theequine epididymis showed that 6 h of culture was sufficient todetect radiolabeled proteins in the media [14]. After 6 h ofculture, media was collected and stored at �80 8C untilconcentrations of lactoferrin were quantified by ELISA.

Lactoferrin concentrations in the media were determinedusing a sandwich ELISA previously described and validated forthe horse [20] and normalized to the weight of the tissue in theculture media. Reagents in the ELISA (coating antibody,alkaline phosphatase conjugated antibody and purified stan-dard) were kindly provided by Dr. Kiyotaka Watanabe (KitasatoUniversity; Japan). The standard curve ranged from 1.5 to100 ng/mL and sensitivity of the assay was 1.56 ng/mL. Intra-assay and inter-assay CV were 5.3% and 10.3%, respectively.

2.4. Statistical analysis

Lactoferrin secretion from epididymal cultures was analyzed byANOVA using the general linear model statement with SASstatistical analysis software (SAS Release 9.2, SAS Institute,Cary, NC, USA). Values for baseline (ng secreted/mg culturedtissue) are reported as least squares means � SEM. Stimulatory

effects of testosterone and estradiol-17b were expressed aspercent change of baseline. Values for hormone effects (%difference) are reported as least squares means � SEM. Signifi-cance of data was determined at p < 0.05 using a two-sided test.

3. Results

Previous studies demonstrated that lactoferrin is produced andsecreted by the mammary gland [17]. Therefore, the antibodywas tested using equine mammary gland tissue to demonstrateits specificity for equine lactoferrin. Positive immunolabeling forlactoferrin was observed in the mammary gland from a lactatingmare (Fig. 1A) but not in the control tissue incubated with goatIgG instead of primary antibody (Fig. 1B). The testis does notexpress lactoferrin [14,30] and immunolabeling of testiculartissue was negative (data not shown). The positive immuno-labeling in the mammary gland, and the lack of expression inthe testis, in conjunction with the lack of immunolabeling innegative control sections (Fig. 2, insets), demonstrates theappropriate specificity of the antibody used for IHC.

Lactoferrin immunolabeling was observed in the cytoplasmof principal cells in the corpus and cauda of peri-pubertal andpost-pubertal stallions (Table 1 and Fig. 2). Expression was

Fig. 2 – Immunolabeling of lactoferrin in the stallion epididymis. Lactoferrin was expressed in the cytoplasm (red color) ofepithelial principal cells (arrows) in the corpus and cauda of peri- and post-pubertal animals as well as on the spermatozoa(S) located in the lumen of the corpus and cauda in post-pubertal animals. Note that principal cells in the caput were negativefor lactoferrin. Left insets: High magnification image of post-pubertal corpus and cauda principal cells. Right insets: negativecontrol. Scale bar: 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of the article.)

r e p r o d u c t i v e b i o l o g y 1 4 ( 2 0 1 4 ) 1 4 8 – 1 5 4 151

particularly strong near the apical membrane of the principalcells, likely the result of vesicle storage near the plasmamembrane prior to secretion. Other cell types of theepididymis were negative in these regions. The caput ofperi-pubertal and post-pubertal animals was negative forlactoferrin. Immunolabeling was not observed in any regionor cell type of the pre-pubertal epididymis (Table 1 and Fig. 2).Sections incubated with goat IgG instead of lactoferrin werenegative (Fig. 2; insets in caput, corpus and cauda of post-pubertal animals). These results suggest that lactoferrinexpression/secretion begins during puberty and continues inthe post-pubertal stage. Sperm in the lumen of the corpus andcauda of post-pubertal animals appeared positive for lacto-ferrin in tissue sections (Table 1 and Fig. 2). To confirm thisobservation, IHC was performed on sperm collected from thecauda of post-pubertal animals. Lactoferrin immunolabelingwas observed on the mid-piece, tail and some sperm heads(Fig. 3).

To investigate the potential regulation of lactoferrinsecretion, tissues from the caput, corpus and cauda wereincubated in vitro with 0, 0.1, 1 or 10 mM of testosterone orestradiol-17b. Results from the 0 dose, shown as the baseline,revealed that the caput secreted 1.14 � 0.81 ng/mg tissue, thecorpus secreted 90.4 � 10.5 ng/mg tissue and the caudasecreted 129.5 � 13.9 ng/mg tissue (Fig. 4). These results areconsistent with the IHC results and demonstrate thatlactoferrin secretion is highest in the cauda. The responseto each dose was similar for testosterone and estradiol.Therefore, values from all doses for each steroid were averagedtogether in the final analysis. Estradiol-17b stimulated asignificant increase in lactoferrin secretion above baseline inthe cauda (Fig. 5). Estradiol-17b appeared to stimulatesecretion in the caput, showing an average increase of 64%above baseline, but the difference was not significant due tothe high variability among animals. The effect of testosteronewas not significant in any region. These data suggest that

Table 1 – Intensity of lactoferrin immunolabeling in thecytoplasm of epithelial principal cells (PC) and sperma-tozoa (S) within the stallion epididymis with respect toage.

Age Caput Corpus Cauda

S PC S PC S PC

Pre-pubertal ND � ND � ND �Peri-pubertal � � +a ++ +a +++Post-pubertal � � ++ +++ +++ +++

Negative (�); weak positive (+); positive (++); strong positive (+++);not determined (ND) since sperm are not present in the epididymisat this age.a When present in peri-pubertal animals.

0

20

40

60

80

100

120

140

160

Caput Corpu s Ca uda

Lact

ofer

rin (n

g/m

g tis

sue)

Epidi dymal Regi on

b

a

b

Fig. 4 – Concentration of lactoferrin in culture mediaincubated for 6 h with the caput, corpus or cauda withouthormone supplementation (baseline). Different superscriptsdesignate significant differences: a and b ( p < 0.05).

r e p r o d u c t i v e b i o l o g y 1 4 ( 2 0 1 4 ) 1 4 8 – 1 5 4152

lactoferrin secretion by the epididymis may be influenced byestradiol, but not testosterone.

4. Discussion

The present results are the first to demonstrate the cellularexpression pattern of lactoferrin in the stallion epididymis andon epididymal sperm. Lactoferrin expression began duringpuberty in the corpus and cauda and was confined to theprincipal cells. The caput did not express lactoferrin at any age.The expression pattern in adult stallions is similar to expressionobserved in other species such as mice and boars [13,29,30].

To verify the immunolabeling results observed in adultstallions, epididymal tissue was cultured to quantify theamount of lactoferrin secreted. Lactoferrin was secreted by thecorpus and cauda with minimal secretion by the caput. Theseresults are in agreement with a previous study for adultstallions demonstrating high levels of lactoferrin secretion bythe corpus and cauda [14]. Additionally, minimal secretion was

Fig. 3 – Immunolabeling of lactoferrin on equine sperm. (A) Spermand tail (arrows). Immunolabeling was periodically observed on

negative controls.

observed from the caput in this study. Immunohistochemistryresults were from the proximal caput, while the culture systemincluded tissue from both proximal and distal caput. Previousresults showed small amounts of secretion from the distalcaput and no secretion from the proximal caput [14].Therefore, our immunolabeling results are consistent withthe secretion data.

Results in the present study also demonstrated that thelactoferrin secreted into the epididymal lumen binds tomature sperm. Lactoferrin was previously localized to the tailof sperm in other species and the results for stallions revealeda similar pattern with binding to the mid-piece and tail.Additionally, some stallion sperm showed binding on thehead. Human sperm were recently demonstrated to express alactoferrin receptor [32] which suggests that lactoferrin bound

were positive for lactoferrin primarily along the mid-piecethe head of sperm. (B) No immunolabeling was observed in

0

20

40

60

80

100

120

Capu t Corpus Cauda

Lact

ofer

rin (%

of b

asel

ine)

Epididymal Regio n

Testosterone

Estra diol

*

Fig. 5 – Hormonal effect(s) on in vitro secretion of lactoferrinin culture media. Bars represent the percent increase inlactoferrin secretion above baseline (seen in Fig. 4) whencultured with testosterone (light gray bars) or estradiol-17b(black bars). * Asterisk indicates a significant increase insecretion above baseline: ( p < 0.05).

r e p r o d u c t i v e b i o l o g y 1 4 ( 2 0 1 4 ) 1 4 8 – 1 5 4 153

to sperm may be a biological event resulting in a role forlactoferrin in protection or regulation of sperm. High con-centrations of lactoferrin were found in the seminal plasma ofhorses [33] and dogs [34]. In both species, seminal lactoferrinwas positively correlated with sperm concentration and totalsperm number. In contrast, high concentrations of lactoferrinin human seminal plasma were found in patients witholigospermia [35] and in semen of horses with low freezability[36]. In the human study, it was suggested that the highconcentrations of lactoferrin in seminal plasma may be due tothe high concentrations of estrogens found in these men [35].In men, serum and testicular estradiol have been shown to beraised in association with oligospermia [37,38]. However, it isunclear as to why an increase in lactorferrin was found inseminal plasma from stallions with low freezability [36] sincelactoferrin is known to have, antimicrobial, antioxidant andimmuno-regulatory properties [39] which would suggest thatlactoferrin is most likely involved in sperm protection.

In the efferent ducts and the initial segment of theepididymis, studies have shown that estrogens regulate fluidreabsorption [22]. In the mouse epididymis, studies haveshown that lactoferrin mRNA is stimulated by estradiol-17b[29]. Estradiol, estrogen conjugates and estrogen receptors arepresent throughout the stallion epididymis [24]; therefore, it ispossible that epididymal lactoferrin in stallions is regulated byestrogens. To test this hypothesis, epididymidal tissue wasincubated with either estradiol-17b or testosterone. Epididy-mal tissue from the cauda cultured with estradiol-17b secretedhigher concentrations of lactoferrin compared to baseline.Tissue cultured with testosterone did not show an increase insecretion. Additionally, it has been demonstrated that theepididymis expresses sulphatase which can convert conjugat-ed estrogens into bioactive estrogens [40,41]. Together, theseresults support the hypothesis that lactoferrin may beestrogen regulated in the stallion epididymis.

The lack of immunolabeling in the pre-pubertal animalsindicates that the expression of lactoferrin initially occursduring puberty. The causes for this up regulation are unclear at

this time, but one possibility is the change in the estrogenenvironment of the equine epididymis. In pre-pubertal stallionsERa was found in the caput and some cells of the corpus but notin the cauda [24]. During puberty, ERa expression increased inthe corpus and cauda such that all principal cells in theseregions were positive. Additionally, estrogen concentrations inthese regions are significantly increased compared to pre-pubertal animals [24]. Together, increases in estrogen concen-tration and receptor expression suggest the importance of thishormone for the pubertal increase in lactoferrin in the equineepididymis. The expression of androgen receptors and testos-terone concentration did not significantly change duringpuberty nor does the expression of ERb [24]. Thus, the increasein lactoferrin could result from the increased signaling ofestradiol-17b acting through ERa.

In conclusion, this study is the first to demonstrate thatlactoferrin is expressed by the principal cells of the corpus andcauda of the equine epididymis, and that this expressionbegins in the peri-pubertal period. Additionally, immunola-beling of caudal sperm from post-pubertal stallions, and theobservation of immunolabeling of sperm in the lumen of thepost-pubertal equine corpus and cauda suggest that lactofer-rin may be necessary for sperm protection. Finally, tissueculture results suggest that epididymal lactoferrin is estrogenregulated in the stallion.

Conflict of interest

None declared.

Role of the funding source

The funding source had no role in the study design; in thecollection, analysis, and interpretation of data; in the writingof the manuscript; or in the decision to submit the manuscriptfor publication.

Acknowledgements

We would like to thank Dr. Kiyotaka Watanabe (KitasatoUniversity; Japan) for kindly providing the ELISA reagents. Wealso thank Lil Sibley for her assistance with the epididymalculture experiments and Jennifer Long for her help inpreparing the discussion. These studies were supported inpart by the Center of Equine Health, with funds provided by theOak Tree Racing Association, the State of California pari-mutual fund, and contributions by private donors.

r e f e r e n c e s

[1] Cooper TG. Role of the epididymis in mediating changes inthe male gamete during maturation. Adv Exp Med Biol1995;377:87–101.

[2] Hammerstedt RH, Parks JE. Changes in sperm surfacesassociated with epididymal transit. J Reprod Fertil Suppl1987;34:133–49.

r e p r o d u c t i v e b i o l o g y 1 4 ( 2 0 1 4 ) 1 4 8 – 1 5 4154

[3] Amann RP. Function of the epididymis in bulls and rams.J Reprod Fertil Suppl 1987;34:115–31.

[4] Glover TD, Nicander L. Some aspects of structure andfunction in the mammalian epididymis. J Reprod FertilSuppl 1971;13(Suppl. 13):39–50.

[5] Robaire B, Hinton B, Orgebin-Crist M-C. The epididymis. In:Neill JD, editor. Knobil and Neill's physiology ofreproduction. 3rd ed., Elsevier; 2006. p. 1071–147.

[6] Briz MD, Bonet S, Pinart B, Egozcue J, Camps R. Comparativestudy of boar sperm coming from the caput, corpus, andcauda regions of the epididymis. J Androl 1995;16:175–88.

[7] Burkin HR, Miller DJ. Zona pellucida protein binding ability ofporcine sperm during epididymal maturation and theacrosome reaction. Dev Biol 2000;222:99–109.

[8] Holtz W, Smidt D. The fertilizing capacity of epididymalspermatozoa in the pig. J Reprod Fertil 1976;46:227–9.

[9] Hunter RH, Holtz W, Henfrey PJ. Epididymal function in theboar in relation to the fertilizing ability of spermatozoa.J Reprod Fertil 1976;46:463–6.

[10] Johnson L, Amann RP, Pickett BW. Maturation of equineepididymal spermatozoa. Am J Vet Res 1980;41:1190–6.

[11] Orgebin-Crist MC, Jahad N. The maturation of rabbitepididymal spermatozoa in organ culture: inhibition byantiandrogens and inhibitors of ribonucleic acid andprotein synthesis. Endocrinology 1978;103:46–53.

[12] Dacheux JL, Belghazi M, Lanson Y, Dacheux F. Humanepididymal secretome and proteome. Mol Cell Endocrinol2006;250:36–42.

[13] Dacheux JL, Belleannee C, Jones R, Labas V, Belghazi M,Guyonnet B, et al. Mammalian epididymal proteome. MolCell Endocrinol 2009;306:45–50.

[14] Fouchecourt S, Metayer S, Locatelli A, Dacheux F, DacheuxJL. Stallion epididymal fluid proteome: qualitative andquantitative characterization; secretion and dynamicchanges of major proteins. Biol Reprod 2000;62:1790–803.

[15] Hall SH, Hamil KG, French FS. Host defense proteins of themale reproductive tract. J Androl 2002;23:585–97.

[16] Lonnerdal B, Iyer S. Lactoferrin: molecular structure andbiological function. Annu Rev Nutr 1995;15:93–110.

[17] Shimazaki K-i, Lactoferrin:. A marvellous protein in milk?J Anim Sci 2000;71:329–47.

[18] Jin YZ, Bannai S, Dacheux F, Dacheux JL, Okamura N. Directevidence for the secretion of lactoferrin and its binding tosperm in the porcine epididymis. Mol Reprod Dev1997;47:490–6.

[19] Wichmann L, Vaalasti A, Vaalasti T, Tuohimaa P.Localization of lactoferrin in the male reproductive tract.Int J Androl 1989;12:179–86.

[20] Inagaki M, Kikuchi M, Orino K, Ohnami Y, Watanabe K.Purification and quantification of lactoferrin in equineseminal plasma. J Vet Med Sci 2002;64:75–7.

[21] Robaire B, Hamzeh M. Androgen action in the epididymis. JAndrol 2011;32:592–9.

[22] Hess RA, Fernandes SA, Gomes GR, Oliveira CA, Lazari MF,Porto CS. Estrogen and its receptors in efferent ductules andepididymis. J Androl 2011;32:600–13.

[23] Stewart BL, Roser JF. Effects of age, season, and fertilitystatus on plasma and intratesticular immunoreactive (IR)inhibin concentrations in stallions. Domest AnimEndocrinol 1998;15:129–39.

[24] Parlevliet JM, Pearl CA, Hess MF, Famula TR, Roser JF.Immunolocalization of estrogen and androgen receptors

and steroid concentrations in the stallion epididymis.Theriogenology 2006;66:755–65.

[25] Kelver ME, Kaul A, Nowicki B, Findley WE, Hutchens TW,Nagamani M. Estrogen regulation of lactoferrin expressionin human endometrium. Am J Reprod Immunol1996;36:243–7.

[26] Pentecost BT, Teng CT. Lactotransferrin is the majorestrogen inducible protein of mouse uterine secretions. JBiol Chem 1987;262:10134–9.

[27] Teng CT, Beard C, Gladwell W. Differential expression andestrogen response of lactoferrin gene in the femalereproductive tract of mouse, rat, and hamster. Biol Reprod2002;67:1439–49.

[28] Syntin P, Dacheux JL, Dacheux F. Postnatal developmentand regulation of proteins secreted in the boar epididymis.Biol Reprod 1999;61:1622–35.

[29] Yu LC, Chen YH. The developmental profile of lactoferrin inmouse epididymis. Biochem J 1993;296(Pt 1):107–11.

[30] Pearl CA, Roser JF. Expression of lactoferrin in the boarepididymis: effects of reduced estrogen. Domest AnimEndocrinol 2008;34:153–9.

[31] Goodman HM. Basic medical endocrinology. New York:Elsevier; 2009: 4–26.

[32] Wang P, Liu B, Wang Z, Niu X, Su S, Zhang W, et al.Characterization of lactoferrin receptor on humanspermatozoa. Reprod Biomed Online 2011;22:155–61.

[33] Kikuchi M, Takao Y, Tokuda N, Ohnami Y, Orino K,Watanabe K. Relationship between seminal plasmalactoferrin and gonadal function in horses. J Vet Med Sci2003;65:1273–4.

[34] Kikuchi M, Mizoroki S, Kubo T, Ohiwa Y, Kubota M, YamadaN, et al. Seminal plasma lactoferrin but not transferrinreflects gonadal function in dogs. J Vet Med Sci 2003;65:679–84.

[35] Buckett WM, Luckas MJ, Gazvani MR, Aird IA, Lewis-JonesDI. Seminal plasma lactoferrin concentrations innormal and abnormal semen samples. J Androl 1997;18:302–4.

[36] Jobim MI, Trein C, Zirkler H, Gregory RM, Sieme H, MattosRC. Two-dimensional polyacrylamide gel electrophoresis ofequine seminal plasma proteins and their relation withsemen freezability. Theriogenology 2011;76:765–71.

[37] Krause W. Concentrations of estradiol in serum and testistissue in patients with fertility disorders. Fertil Steril1988;49:926–7.

[38] Levalle OA, Zylbersztein C, Aszpis S, Mariani V, Ponzio R,Aranda C, et al. Serum luteinizing hormone pulsatility andintratesticular testosterone and oestradiol concentrationsin idiopathic infertile men with high and normal folliclestimulating hormone serum concentrations. Hum Reprod1994;9:781–7.

[39] Gonzalez-Chavez SA, Arevalo-Gallegos S, Rascon-Cruz Q.Lactoferrin: structure, function and applications. Int JAntimicrob Agents 2009;33(301):e1–8.

[40] Lemazurier E, Seralini GE. Evidence for sulfatase and17beta-hydroxysteroid dehydrogenase type 1 activities inequine epididymis and uterus. Theriogenology 2002;58:113–21.

[41] Zdunczyk S, Janowski T, Ras A, Baranski W. Activity ofsteroid sulphatase and estrogen sulphotransferase in theboar epididymis during the postpubertal period. Reprod Biol2012;12:374–8.