Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes

Accepted Manuscript Title: Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes Authors: Marcelo D. Goissis, Mariana I. Giassetti, Robinson A. Worst, Camilla M. Mendes, Pedro V. Moreira, Mayra E.O.A. Assumpc¸a˜ o, Jose A. Visintin PII: DOI: Reference:

S0378-4320(18)30252-5 https://doi.org/10.1016/j.anireprosci.2018.08.014 ANIREP 5916

To appear in:

Animal Reproduction Science

Received date: Revised date: Accepted date:

14-3-2018 27-7-2018 10-8-2018

Please cite this article as: Goissis MD, Giassetti MI, Worst RA, Mendes CM, Moreira PV, Assumpc¸a˜ o MEOA, Visintin JA, Spermatogonial stem cell potential of CXCR4positive cells from prepubertal bull testes, Animal Reproduction Science (2018), https://doi.org/10.1016/j.anireprosci.2018.08.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes

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Marcelo D. Goissis, Mariana I. Giassetti1, Robinson A. Worst, Camilla M. Mendes, Pedro V. Moreira, Mayra E. O. A. Assumpção, Jose A. Visintin

Department of Animal Reproduction, School of Veterinary Medicine and Animal

author: Marcelo D. Goissis; Department of Animal Reproduction Av.

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*Corresponding

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Science, University of São Paulo, Brazil

address: School of Molecular Biosciences, College of Veterinary Medicine,

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1Current

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Orlando Marques de Paiva, 87, São Paulo, SP, Brazil 05508-070; [email protected]

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Washington State University, U.S.

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Highlights for Goissis et al. "Spermatogonial stem cell potential of CXCR4-

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positive cells from pre-pubertal bull testis"

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

CXCR4 is expressed in few cells of the pre-pubertal bovine testis



CXCR4-enriched cell population present expression of spermatogonial markers



Xenotransplantation of CXCR4-enriched cells yield more colonies in mouse testis

ABSTRACT Spermatogonial stem cells (SSC) have the potential to restore spermatogenesis when

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transplanted into testes depleted of germ cells. Due to this property, SSC could be used in breeding programs and in transgenic animal research. Particularly in cattle, SSC are not as well characterized as in mice or humans. In mice, C-X-C Motif Chemokine Receptor 4 positive (CXCR4+) testicular cells have high SSC potential. It, therefore, was hypothesized that CXCR4 is a marker of undifferentiated spermatogonia in cattle.

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Using samples from pre-pubertal calves, the CXCR4 protein was detected by

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immunohistochemistry in a few cells of the seminiferous tubules. Testicular cells were

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isolated, frozen-thawed and submitted to magnetic-activated cell sorting using anti-

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CXCR4 antibody. Quantitative RT-PCR analysis revealed that CXCR4+ cells had THY1,

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OCT4 and ZBTB16 (or PLZF) mRNA in these cells. Flow cytometry results indicated

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that the proportion of THY1+ cells is enriched in CXCR4+ populations. Colonization potential of CXCR4+ cells was assessed after xenotransplantation into testes of nude

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mice treated with busulfan. Transplantation of CXCR4+ cells yielded an increase of 5.4-

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fold when compared to CXCR4- cells. These results indicate that CXCR4 could be used as a marker to enrich and sort cells of bulls with putative spermatogonial stem cell

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potential.

Keywords: Spermatogonia; Stem cell; Testicle; Bovine

1. Introduction

stem

cells

(SSC)

are

responsible

for

the

continuous

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Spermatogonial

spermatogenesis throughout the male adult life. It is estimated that only 0.03% of the total testicular cells of an adult animal are SSC ( Tegelenbosch and de Rooij, 1993). The SSC have the potential to colonize and restore spermatogenesis when transplanted into testes depleted of germ cells (Brinster and Avarbock, 1994; Brinster and

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Zimmermann, 1994). This property would allow clinical or biotechnological applications

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of SSC, such as recovery of the germ cell population or generation of transgenic

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livestock (Brinster, 2007, Zeng et al., 2012). Particularly in cattle, some studies have

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identified markers or developed methods for enrichment of spermatogonia ( Herrid et

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al., 2007; Herrid et al., 2009; Reding et al., 2010; McMillan et al., 2014; Giassetti et al.,

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2016) however, SSC are not as well characterized as in mice or humans. More studies, therefore, are required to improve the isolation and further characterize the molecular

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components of bull SSC.

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Isolation and enrichment of SSC from a heterogeneous population of testicular cells are essential for proper characterization of these cells (Phillips et al., 2010;

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Shinohara et al., 1999). In mice, strategies such as differential plating with laminin or gelatin pre-coated dish (Shinohara et al., 1999) or fluorescent-activated cell sorting (FACS) are used for enriching SSC. Several membrane molecules have been described as molecular markers of mouse SSC for use in FACS enrichment of live cells, such as Integrin Alpha-6 (ITGA6; Shinohara et al., 1999), Thy1-antigen (THY1; Kubota et al.,

2003), CD9 (Kanatsu-Shinohara et al., 2004), Glial Cell Line-Derived Neurotrophic Factor Receptor alpha-1 (GFRa1; Buageaw et al., 2005; Hofmann et al., 2005). No

SSC in domestic animals (Phillips et al., 2010).

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individual marker or a combination of markers, however, allows for isolation of pure

In cattle, isolation of putative spermatogonial stem cells was previously performed using differential plating and colonization potential was verified by affinity to Dolichos biflorus a)gglutinin or Ubiquitin C-Terminal Hydrolase L1 (UCHL1) staining (Izadyar et al., 2002; Herrid et al., 2009 ). A more specific approach was demonstrated magnetic-activated

cell

sorting

(MACS)

using

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after

THY1

antibody

and

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xenotransplantation of enriched cells into mouse testes (Reding et al., 2010). Cells

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expressing C-X-C Motif Chemokine Receptor 4 (CXCR4+ cells) were capable of

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restoring spermatogenesis after functional transplantatioAn in mice (Yang et al., 2013).

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Knockout or knockdown of CXCR4 impaired the SSC potential of transplanted cells

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(Kanatsu-Shinohara et al., 2012; Yang et al., 2013). The CXCR4 protein is the receptor for chemokine CXCL12 and this signaling duo is associated with hematopoietic stem

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2003).

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cell migration (Peled et al., 1999) or primordial germ cell migration (Molyneaux et al.,

The CXCR4-CXCL12 signaling is postulated to be important in migration of SSC

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to the stem cell niche (Kanatsu-shinohara and Shinohara, 2013), which could increase efficiency of cell transplantation. CXCR4+ cells are contained within the THY1+ cell population and may represent the stem cell fraction within a progenitor cell pool (Yang et al., 2013). Based on this information, it is hypothesized that CXCR4 is a marker of undifferentiated spermatogonia in bulls. To test this hypothesis, the expression of the

CXCR4 gene in prepubertal testicular cells was examined followed by enrichment of CXCR4+ cells through use of MACS. The enriched cells were characterized by gene expression and protein presence evaluations and assessed colonization potential by

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xenotransplantation into testes of immunocompromised mice.

2. Material and methods

All reagents were purchased from Sigma-Aldrich (St. Louis, MO, EUA) unless

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otherwise stated.

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2.1. Animal use

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Animal use during this experiment was approved by the Ethics Committee on

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Use of Animals of the School of Veterinary Medicine and Animal Science of the

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University of Sao Paulo (CEUAVET 2809/2012). Nellore bull calves were maintained in pasture along with their dams with water ad libitum in the Campus Fernando Costa of

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the University of São Paulo, in Pirassununga, SP. Calves were transported to the

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Department of Animal Reproduction facilities 1 day prior to orchiectomy and restricted of water and food for 24 h prior to surgery. The Balb/c nude mice were housed in mini-

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isolators on a normal 12-hour light/dark cycle with ad libitum food and water.

2.2. Experimental Design

Testicular tissue from prepubertal bull calves (n = 8) was obtained after orchiectomy. Samples were used in immunocytochemistry to detect the CXCR4 protein and spermatogonial marker UCHL1. Isolated cells were frozen and then thawed for

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experiments. Flow cytometry was used initially to verify the proportion of cells that were CXCR4+. Magnetic-activated cell sorting (MACS) was performed to separate CXCR4+ and CXCR4- cells and results were confirmed by flow cytometry. Cells were molecularly characterized after MACS by examining gene expression of commonly used spermatogonial or germline markers by quantitative RT-PCR (qPCR, n=5) and by

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assessing presence of GFRA1+ or THY1+ cells using flow cytometry (n = 5). A

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xenotransplantation assay of CXCR4+ and CXCR4- cells was performed using

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immunocompromised nude mice to verify stem cell potential of sorted cells from eight

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different bull calves. Due to procedure failure, one bull was excluded from the

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xenotransplantation experiment, reducing the experimental number (n = 7).

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2.3. Tissue and cell collection

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Testicles were obtained from 5 to 6 month old Nellore (Bos taurus indicus) bull calves after surgical orchiectomy. A total of eight calves were used for this experiment.

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Testes were dissected and fragments of 1cm 3 were fixed in paraformaldehyde 4% for 24 h and transferred to 95% ethanol solution until embedded in paraffin. The remaining testicular tissue (approximately 10 g) was minced and then incubated for 30 minutes at 37 °C with agitation in 30 ml of collagenase solution containing 1 mg/ml collagenase V, 5 µg/ml DNAse, 40 µg/ml gentamycin, 100 UI/ml penicillin and 1% non-essential amino

acids (NEAA;v/v) in DMEM medium (Life Technologies, Carlsbad, CA, USA). Digested seminiferous tubules were washed three times with PBS (Life Technologies) at 50 x g for 1 minute. Next, samples were incubated for 5 minutes at 37 °C with agitation in 30

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ml of trypsin solution containing 2.5 mg/ml trypsin, 0.82 mg/ml EDTA, 5 µg/ml DNAse, 40 µg/ml gentamicin, 100 UI/ml penicillin and 1% NEAA (v/v) in DMEM medium. Trypsin was inactivated with 10% (v/v) FBS (Life Technologies) and samples were homogenized by pipetting. Cellular suspension was filtered through a 100 µm nylon mesh (Falcon BD, Franklin Lakes, NJ, USA) and centrifuged at 600 x g for 8 minutes.

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Cells were re-suspended in 2 ml of PBS and 10 µl were stained with Trypan Blue (Life

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Technologies). Cells were centrifuged at 600 x g for 5 minutes and then re-suspended

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in freezing medium containing 10% FBS (v/v), 10% DMSO (v/v) and 0.07M sucrose in

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DMEM (Izadyar et al., 2002). Cryovials containing 10 x 106 cells were allocated to a

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freezing container (Mr. Frosty, Nalgene, Waltham, Massachusetts, USA) during 24 h

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and then placed in liquid nitrogen until thawing.

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2.4. Immunohistochemistry

Immunohistochemistry was performed to verify the presence of cells expressing

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CXCR4 in the testis. Paraffin embedded testicular tissues were cut in 5 µm thick slices and placed onto silanized glass slides. Paraffin was removed and tissue was rehydrated after sequential decreasing concentration of xylene baths followed by ethanol baths. Slides were washed in water and submitted to antigen retrieval for 20 minutes in microwave with Target Retrieval Solution (Dako, Agilent Technologies, Santa Clara, CA,

USA).

Peroxidase blocking was performed with incubation for 30 min at room

temperature with 1.5% (v/v) hydrogen peroxidase in water. Nonspecific binding sites were blocked by 10% FBS (v/v) solution in PBS for 1 h at room temperature. Antibodies

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against CXCR4 (1:250, ab7199 Abcam Cambridge, MA, USA) or spermatogonial marker UCHL1 (1:100, ab72911, Abcam) were used as primary antibodies. Primary antibody was incubated with samples overnight at 4 °C. Samples incubated in 10% FBS solution in PBS were used without primary antibody as a control (Figure 1C). Slides were then washed with water and Advance HRP Kit (Dako) was used for staining

with

haematoxylin

for 20

seconds and

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incubated

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according to manufacturer’s instructions. After peroxidase staining slides were dehydrated in

increasing

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concentrations of ethanol and xylene baths. Coverslips were placed with Permount

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(Thermo Scientific, Waltham, MA, USA) and samples were then imaged at an inverted

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IX81 Olympus microscope. Images were obtained using software Image-Pro Plus

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(Media Cybernetics, Rockville, MD, USA).

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2.5. Magnetic-activated cell sorting (MACS)

Enrichment of CXCR4+ cells was performed using MACS (Miltenyi-Biotec,

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Teterow, Germany). Cells were thawed in DMEM (Life Technologies) supplemented with 30 mg/ml of BSA and then re-suspended in PBS with 10% FBS. OctoMACS Separator and MS-columns (Miltenyi-Biotec) were used according to manufacturer’s instructions. Anti-CXCR4 (1:500, ab7199, Abcam) was used as primary antibody and MACS anti-rabbit IgG (Miltenyi-Biotec) as secondary antibody. Cells collected in the

flow through were considered negative for CXCR4 (CXCR4-) and cells pushed after magnetic selection were considered positive for CXCR4 (CXCR4+). Both cellular

cytometry, quantitative PCR and xenotransplantation.

2.6. Western blotting

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populations were used for downstream applications such as western blotting, flow

Proteins were extracted from cells immediately after MACS sorting using RIPA

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lysis buffer supplemented with 1% (v/v) protease inhibitor cocktail. Samples were

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sonicated for 30 s and total protein amount was determined using Qubit Protein Assay

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kit and Qubit fluorometer (Life Technologies). Novex (Life Technologies) LDS Sample

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buffer and Bolt Reducing Agent were then added to 80 μg of protein according to

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manufacturer's instructions. Samples were then incubated at 70 °C for 10 minutes and

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applied on Novex 12% Bis-Tris plus gels (Life Technologies) using 200 V for 22 minutes.

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Proteins were transferred from gel to nitrocellulose membranes using iBlot2 Gel

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Transfer Device and iBlot® 2 Transfer Stacks (Life Technologies) according to manufacturer's instructions. Membrane was then blocked with a Tris-NaCl solution

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supplemented with 1% (v/v) Tween-20 (TBS-T) and 5% (w/v) of bovine serum albumin for 1 h at room temperature. Membranes were incubated with anti-CXCR4 (1:2000, ab7199, Abcam) or antitubulin (1:500, ab125267, Abcam) antibodies for 16 h at 4 °C with constant agitation. Next, we washed membranes with TBS-T four times for 5 minutes at room temperature.

Secondary antibody anti-rabbit-HRP (1:10000, ab97051, Abcam) was added for 1 h at room temperature. Membranes were washed again in TBS-T as above. SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) was

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used according to manufacturer's instructions and chemoluminescent signal was observed using ChemiDoc system (BioRad, Hercules, CA, USA).

2.7. Flow cytometry

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The population of CXCR4+ cells within isolated testicular cells was determined

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by flow cytometry. Cells were thawed as described above and incubated with anti-

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CXCR4 (1:500, ab7199, Abcam) for 30 minutes at room temperature in PBS

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supplemented with 10% (v/v) FBS. Cells were washed with PBS for 3 minutes at 950 x

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g. A FITC conjugated anti-rabbit (1:200, ab97050, Abcam) was used as secondary

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antibody and incubated with cells for 30 minutes at room temperature in PBS supplemented with 3% (v/w) BSA. Cells were washed with PBS for 3 minutes at 950 x

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g prior to flow cytometry using FACScalibur (Becton Dickinson Immunocytometry

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Systems, San Jose, CA, USA). Blank cells (no antibody) and cells incubated only with secondary antibody (control) were used as controls.

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To assess CXCR4+ population after MACS (n = 8), a FITC conjugated anti-rabbit

(1:200, ab97050, Abcam) was used as secondary antibody and incubated for 20 minutes at 4 °C along with the MACS anti-rabbit IgG. Washings were performed as indicated by MACS separation protocol. Both CXCR4- and CXCR4+ cell fractions were submitted to flow cytometry. Blank unsorted cells (no antibody), unsorted cells

incubated only with secondary antibody and unsorted cell samples were used as control. Verification of GFRA1 positive (GFRA1+) cells was performed after enrichment of

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CXCR4+ cells through MACS (n = 5). Unsorted, CXCR4+ and CXCR4- cells were centrifuged and then incubated for 30 minutes in a 25 mM glycine solution made with PBS supplemented with 3% BSA and pH adjusted to 2.7 to remove surface bound antibodies as described previously (Xiao et al., 2003). A rabbit anti-GFRA1 antibody (1:500, ab84106, Abcam) was used as a primary antibody and a goat FITC conjugated

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anti-rabbit antibody was used as secondary. Verification of THY1 positive (THY1+) cells

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was performed along with enrichment of CXCR4+ cells through MACS (n=5). A mouse

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anti-THY1 antibody (1:20, ab23894, Abcam) was used as primary antibody

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simultaneously with rabbit anti-CXCR4 antibody used for MACS. A goat FITC

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conjugated anti-mouse was used as secondary antibody, concomitantly to the MACS

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anti-rabbit IgG incubation. Blank unsorted cells (no antibody), unsorted cells incubated only with secondary antibody and unsorted cell samples were used as control.

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Data were interpreted using FlowJo software (TreeStar Data Analysis Software,

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Ashland, OR, USA). Staining with 50 µg/ml of propidium iodide was used to identify cell population within overall events. Threshold numbers of positive cells were determined

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based on the blank cell samples. Background values were determined based on the percentage of positive events above the threshold level in samples incubated with secondary antibody only. Background values were then subtracted from the percentage of positive cells in unsorted or sorted cells.

2.8. Quantitative RT-PCR

Quantitative PCR was used to characterize the relative abundance of mRNA

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commonly associated with undifferentiated spermatogonia. The RNA was isolated from CXCR4- and CXCR4+ cell samples (n = 5) using PicoPure RNA Isolation Kit (Life Technologies) including the DNase incubation step (Qiagen, Venlo, Netherlands). RNA was quantified using Nanodrop spectrophotometer (Thermo Scientific). Reverse transcription was performed using SuperScript VILO™ cDNA Synthesis Kit (Life

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Technologies). The resulting cDNA was quantified with Qubit dsDNA BR Assays kit

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(Life Technologies) and Qubit 2.0 Fluorometer (Life Technologies). Samples were

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normalized to 200 ng/ml of cDNA prior to PCR reactions. Quantitative PCR was

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conducted using SYBR GreenER™ qPCR Supermix Universal (Life Technologies) at

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the following program: 2 min at 50 ºC, 10 min at 95 ºC, 40 cycles at 95 ºC for 15 s and

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60 ºC for 1 min, plus a melting curve step (Mastercycler Ep Realplex Thermal Cycler, Eppendorf AG, Hamburg, Germany). Verification occurred regarding the expression of

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genes related to spermatogonial cells and pluripotency: GFRA1, THY1, ZBTB16,

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ITGA6, UCHL1, VASA, OCT4, SOX2 and NANOG. Primers were designed using online software Primer Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi), considering optimal

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melting temperature of 60 °C and maximum and preferentially spanning one intron. Product sizes ranged from 170 to 200 bp. Primers and concentrations used in this study are described in table 1. Primers efficiencies were determined by use of a standard curve and optimal efficiencies were considered between 0.9 and 1.0. Primers with lesser efficiencies were discarded and, therefore, not listed in Table 1. Geometric mean

of housekeeping genes ACTB and GAPDH was used to normalize the relative quantification of target genes. Housekeeping genes were previously selected (De Barros et al., 2012) using Normfinder (Andersen et al., 2004). Quantification cycle (Cq)

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average values are listed in Table 1.

2.9. Xenotransplantation into mouse testis

Xenotransplantation assays were performed to assess stem cell potential of

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CXCR4+ cells. Balb/c nude mice at 31-days of age that were treated with 44 mg/kg

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busulfan IP to deplete germ cells. Xenotransplantation assays were done after 40 to 44

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days after busulfan treatment. At the day of transplantation, mice were treated with 4

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mg/kg morphine SC (Hipolabor, Belo Horizonte, MG, Brazil) and anesthetized using

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isoflurane (Biochimico, Itatiaia, RJ, Brazil). Transplantation was performed as described

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previously (Reding et al., 2010; Tang et al., 2012). Briefly, a 1 cm incision was made to expose the testis and epididymis. The efferent ducts were isolated and cannulated

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using a fine glass pipette for cell injection.

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A total of eight mice received cells from eight different bull calves. Each mouse received cells from one bull calf. CXCR4- cells were injected in the left testicle while

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CXCR4+ cells were injected in the right testicle. Injection failed in one mouse and seven mice were considered in the experiment (n = 7). The CXCR4- and CXCR4+ cells obtained after MACS enrichment were stained with red fluorescent cell linker PKH26 following manufacturer’s instructions. Cells were re-suspended in PBS at a concentration of 1 x 103 cells/µl. A solution of 0.4% Trypan blue was added at 20% (v/v)

to track the success of the procedure. Injected volume was measured based on the volume loaded in the pipette and the remaining volume after injection. Injected volume was used to determine the number of injected cells in each testis.

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Mice were euthanized and cellular colonization was verified after 28 days of the transplant. Testes were dissected and incubated in 1 mg/ml collagenase in PBS for dissociation of seminiferous tubules. Tubules were washed three times with PBS and were entirely spread onto 35 mm petri dishes for visualization under Olympus IX-81 epifluorescence microscope with 550 nm excitation filter and 590 nm emission filter.

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Only regions containing cellular chains were counted as positive colonization. Three

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individuals did colonization analysis; colonies were only counted if all observers

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considered the same region as a colony. Single or double stained cells were not

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counted as colonies. Colony number was normalized by 1 x 105-injected cells in each

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testis.

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2.10. Statistical analysis

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Data were analyzed using SAS 9.3 software (SAS Institute, Cary, NC, USA). In FC and qPCR experiments, dependent variable was the percentage of CXCR4+ cells

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and independent variable was the fraction of MACS sorted cells (flow through compared with positive selection). In xenotransplantation experiments, dependent variable was the number of colonies and independent variables were the fraction of MACS sorted cells and donor bull, which was considered as a random effect. The FC and xenotransplantation data were submitted to PROC UNIVARIATE of SAS to assess

normality and homogeneity of variances. The FC data were then transformed by Log10 and analyzed using PROC GLM of SAS with Tukey’s post hoc test for comparison of means. PCR data were analyzed using PROC MIXED of SAS as described (Steibel et

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al., 2009). The PCR data are presented in log2 scale in figures while fold change is used in the text for ease of interpretation. Xenotransplantation data were analyzed using PROC MIXED of SAS with Tukey’s post hoc test for comparison of means. Significant statistical difference was considered if P≤0.05.

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3. Results

CXCR4+

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detect

cells

in

the

testis

or

isolated

testicular

cells,

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To

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3.1. Detection of CXCR4+ cells

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immunohistochemistry (IHC) and flow cytometry (FC) analysis were performed using anti-CXCR4 antibody. Testicular tissue from prepubertal bull calves was submitted to

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IHC and few cells of the seminiferous tubules were positive for CXCR4 staining (Figure

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1A), while more cells were positive for spermatogonial marker UCHL1 (Figure 1B). Isolated testicular cells were submitted to FC analysis and an average of 1.87% of the

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total cell population was observed to be CXCR4+ (Figure 1D and 1E).

3.2. Magnetic activated cell sorting

To enrich CXCR4+ cells from the total cell population MACS was used. Flow cytometry analysis was used to assess efficiency of the MACS procedure. Cells obtained in the flow through (CXCR4-) and positively selected cells (CXCR4+) were

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used for FC analysis. Use of MACS allowed for enrichment of CXCR4+ cells as an average of 29.2% of MACS sorted cells were CXCR4+ (Figure 2A and 2B), MACS yielded a significant 15.52-fold enrichment of CXCR4+ cells when compared to unsorted cells and a 21.99-fold enrichment when compared to flow through CXCR4- cells (Figure 2B). Using western blotting with anti-CXCR4 antibody, a single ~50KDa protein band

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both CXCR4- and CXCR+ samples (Figure 2C).

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was detected only in the CXCR4+ positive samples, while alpha-tubulin was detected in

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3.3. Molecular characterization of CXCR4+ cells

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The CXCR4+ and CXCR4- cells sorted by MACS were used for RNA extraction and subsequent q-RT-PCR. Relative abundance quantification was performed for

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GFRA1, THY1, ZBTB16, ITGA6, UCHL1, VASA, OCT4, SOX2 and NANOG. NANOG

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mRNA was not detected in any of the samples. Genes that were upregulated in the CXCR4+ cell population when compared to CXCR4- cells (Figure 3) included THY1

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(3.94-fold), ZBTB16 (7.49 fold) and OCT4 (2.69-fold). The other genes such as GFRA1, ITGA6, UCHL1, VASA and SOX2 were not differentially expressed (Figure 3). In addition to gene expression, the presence of GFRA1 positive (GFRA1+) and THY1 positive (THY1+) cells was verified within the CXCR4+ population after MACS using anti-CXCR4 antibody. Flow cytometry analysis revealed that the percentage of

GFRA1+ cells was not different among unsorted cells, CXCR4- and CXCR4+ cells (Figure 4A and 4B). Percentage of THY1+ positive cells, however, was significantly

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enriched within the CXCR4+ cell population (Figure 4C and 4D).

3.4. Xenotransplantation of CXCR4+ cells

To assess stem cell potential of CXCR4+ cells MACS sorted cells were transplanted into testis of nude mice treated with busulfan. After 28 days of

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transplantation the efficiency of colony formation was verified. Only areas were

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considered with intense colonization after tubule dissociation as previously described in

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this manuscript (Figure 5A). There was a larger number of colonies in testes injected

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with CXCR4+ cells when compared to CXCR4- injections (Figure 5A and 5B). Colony

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formation was enriched by 5.40-fold (P = 0.017) when considering the colony number

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per 105 injected cells (Figure 5C). Mean colony number per 105 injected CXCR4- cells was 12, 83 with a 95% confidence interval from 12, 54 to 13,12. Mean colony number

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70, 92.

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per 105 injected CXCR4+ cells was 69.4 with a 95% confidence interval from 67, 81 to

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5. Discussion

In the present study, there was evaluation of the stem cell potential of bull

spermatogonia that are positive for CXCR4 protein. Spermatogonial stem cells (SSC) have the potential to colonize testis depleted of germ cells and reinitiate spermatogenesis (Brinster and Avarbock, 1994; Brinster and Zimmermann, 1994). This

property would allow the use of SSC in animal reproductive biotechnologies, including animal transgenesis. Selection of more undifferentiated cells is necessary to increase transplantation efficiency into recipient testis (Brinster, 2007).

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Immunohistochemistry images in the present study indicate that some CXCR4+ and UCHL1+ cells were not at the basal lamina, suggesting that CXCR4+ cells could be migrating gonocytes. Staining with ZBTB16 could have provided stronger evidence that CXCR4 cells are indeed gonocytes, still, both CXCR4+ and UCHL1 cells had similar morphology, comprising larger cells, with large nuclear/cytoplasmic ratio, suggesting

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both UCHL1 and CXCR4 mark gonocytes. In addition, no differences in gene

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expression were observed between UCHL1 and ZBTB16 (PLZF) in bull gonocytes (Cai

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et al., 2016). In Brahman bulls, gonocytes were fully observed at the basal lamina of the

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seminiferous tubule at 8.5 months of age (Aponte et al., 2005), while in the present

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study 5 to 6 month old Nellore calves were used. Gonocytes were shown to have stem

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cell potential as demonstrated by transplantation assays (Orwig et al., 2002). Immunohistochemistry analysis revealed that CXCR4 protein is present in fewer

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putative gonocytes while a larger number of gonocytes were positive for UCHL1

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staining, suggesting that CXCR4+ cells are contained within UCHL1+ cells. In mice, CXCR4 was detected in a subgroup of cells that contained the spermatogonial cell

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marker, ZBTB16 (Yang et al., 2013). Additionally, CXCR4+ cells were contained in the THY1+ cell population (Yang et al., 2013), which was previously shown to have stem cell potential in bulls (Reding et al., 2010). In bulls, THY1 was localized in a subset of cells expressing ZBTB16, which in turn was present in fewer cells than UCHL1 when evaluating prepubertal bull testis (Reding et al., 2010). Because CXCR4 was observed

to be in less cells than UCHL1, it was speculated that these were a subpopulation of cells with spermatogonial potential in bulls and subsequently enrichment and transplantation experiments were performed.

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The MACS utilization allowed the enrichment of CXCR4+ cells by 22.0-fold, enabling downstream analysis of these cells such as qPCR. The THY1, ZBTB16 and OCT4 genes were expressed and expression was greater in the CXCR4+ cell population, possibly indicating the stem cell potential of these cells. For instance, THY1 is used as marker of mice, non-human primate and cattle SSC ( Kubota et al., 2003;

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Hermann et al., 2009; Reding et al., 2010). In addition to increased gene expression,

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the CXCR4+ cell population was observed to be enriched for THY1+ cells, further

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supporting the idea that these cells hold spermatogonial stem cell potential. Likewise,

M

ZBTB16 (PLZF) is essential for self-renewal (Costoya et al., 2004) and is co-expressed

D

with OCT4 (Buaas et al., 2004) in mice while it can also be used to identify

TE

undifferentiated spermatogonia in cattle (Reding et al., 2010) and goats (Abbasi et al., 2013). Recently, it was shown that ZBTB16 is involved in the regulation of CXCR4 gene

EP

expression through modulation of miRNA in goat SSC (Mu et al., 2015). Lastly, OCT4 is

CC

expressed in mice SSC (Dann et al., 2008) and OCT4 has been used in mice SSC selection by fluorescence-activated cell sorting (Guan et al., 2009). Noteworthy, amount

A

of gene expression was not consistent with cellular enrichment and there was not evaluation of specific Sertoli cell markers in the present study, thus more research is required to refine the isolation of CXCR4+ cells. Surprisingly, gene expression of other SSC markers such as GFRA1, VASA and ITGA6 were unchanged in CXCR4+ and CXCR4- cell populations in contrast to what

was previously observed in cells enriched through CXCR4 or other markers (Hermann et al., 2009; Reding et al., 2010; Yang et al., 2013). In agreement with the gene expression data, the percentage of GFRA1+ cells was not increased after MACS sorting

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of CXCR4+ cells. The GFRA1 is accepted as a marker of murine spermatogonial stem cells ( Meng et al., 2000; Hofmann et al., 2005) and is required for its self-renewal (He et al., 2007). The GFRA1 was also reported to be expressed in cells of immature mouse testis (Buageaw et al., 2005). Recently, GFRA1 was detected in gonocytes of 1-day-old (Cai et al., 2016) and 3-month-old (Suyatno et al., 2018) bulls. This fact along with

U

observations in the present study suggest that GFRA1 may be a marker for most germ

N

cells, while CXCR4+ could only be a marker for a subset of GFRA1+ cells.

A

The lack of difference in gene expression of VASA could also result from

M

enrichment through MACS, which may not sort all germ cells, but only those which

D

express CXCR4. In cattle VASA is present in most of the UCHL1+ cells (Fujihara et al.,

TE

2011), thus the CXCR4- fraction could contain germ cells which express the VASA gene. The ITGA6 gene is also expressed by Sertoli cells in rats (Beardsley et al., 2006),

EP

which would possibly explain why gene expression of ITGA6 is not different between

CC

CXCR4+ and CXCR4- fractions. Transplantation

of

CXCR4+

cells

into

germ

cell-depleted

testis

of

A

immunocompromised mice yielded more colonies than transplantation of CXCR4- cells, which indicates there was a larger number of stem cells in the positively selected cell population. Colony formation was increased by approximately 5-fold, similarly to what was previously observed in transplantation of THY1+ cells in cattle and goats ( Reding et al.,

2010; Abbasi et al., 2013) and similar to mouse cells selected by CXCR4 (Yang et al., 2013). These data, however, cannot assure that the CXCR4+ marker in cattle represents the stem cell population in the testis. Direct comparison between THY1+ and

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CXCR4+ cells should be performed to assess which cell population is more efficient to generate colonies after transplantation. Furthermore, bull Sertoli cells are also able to colonize mouse testis (Zhang et al., 2008) and absence of these cells should be ensured in future experiments.

In summary, in the present study a small subset of cells in the bull prepubertal

U

testes contain the CXCR4 protein and can be selected through MACS. These cells have

N

greater expression of THY1, OCT4 and ZBTB16 genes than CXCR4- cells.

A

Furthermore, the THY1+ cell population is enriched after CXCR4 sorting; and CXCR4+

M

were able to colonize testis after 28 days and establish 5-fold more colonies than

EP

6. Conclusion

TE

D

CXCR4- cells.

CC

The CXCR4 could be used as a marker to enrich frozen-thawed bull pre-pubertal testicular cells with spermatogonial stem cell potential. In the future, these cells could be

A

used in breeding programs and for generation of transgenic livestock.

Disclosure Authors declare that there is no conflict of interest. All authors have approved the final version of this manuscript. MDG designed the study, performed experiments, analyzed

data, interpreted data and drafted the final version of the manuscript. MIG, RAW and CMM performed orchiectomy, xenotransplantation assays and analyzed data. PVM

designed the study and drafted the manuscript.

Funding

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performed orchiectomy, qPCR experiments and analyzed data. MEOAA and JAV

This research was funded by Sao Paulo Research Foundation (FAPESP), grant

Conflict of interest statement

M

A

Authors declare that there is no conflict of interest.

N

U

2012/16157-3 to MDG and grant 2013/03495-0 to JAV.

D

Acknowledgements

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Authors would like to thank Prefeitura do Campus de Pirassununga for providing the bull calves used in this experiment; Prof. Paulo Maiorka in the Department of Pathology

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for assistance with sample preparation for immunohistochemistry; Prof.Cristina de Oliveira Massoco Salles Gomes and Nicolle Queiroz in the Department of Pathology for

CC

assistance with flow cytometry; Prof. Paula Papa and Dr.Renata Silva for assistance with sonication of samples and ChemiDoc, Carolina D. N. Pereira for assistance with

A

mouse anesthesia; and Prof. Ricardo J. G. Pereira for critical reading of the manuscript.

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Tables

Table 1: Primer sequences used for qPCR in the present study along with respective concentrations and observed quantification cycles

GFRA1

M

CC E

THY1

ED

GAPDH

F - GTCCACCTTCCAGCAGATGT R - GTCACCTTCACCGTTCCAGT F - TGACCCCTTCATTGACCTTC R - TACTCAGCACCAGCATCACC F - CAAGTGGAGCACATCTCGAA R - GGCAGGAACAGAAGAGCATC F - TGCTAACAGTCTTACAGGTGGC R - TCTTTGTGTCACGGGTCAGG F - TACCGCCGTGATACCGAGAG R - CCGAGGCTTAGGCATGAGG F – AGGGATGTGGAGACGACAAC R - TCTTTGGTGGGATTCTTTGG F - TGCTGAACAAAGTGCTGACC R - GATGATGGAACCGAGATGCT F - TTTGCCTCTGGGAGGAGTTTG R - TTGGAAAACCCTCTGTTCCGT F - TACTGTGCGCCGCAGGTTGG R - GCTTTGATGTCCTGGGACTCCTCA F - AGCGCATGGACAGCTACGCG

PT

ACTB

Primer sequence (5’ to 3’)

ZBTB16

A

ITGA6

UCHL1 VASA OCT4 SOX2

A

GenBank

Gene

Reference NM_173979 NM_001034034.2 NM_001105411 NM_001034765 NM_001037476.2 NM_001109981 NM_001046172.2 NM_001007819.1 NM_174580 NM_001105463

Concentration in Average Cq values

reaction (nM) 200 200 200 400 200 200 200 200 200 200 200 400 200 200 200 200 300 300 200

20,58 22,92 29,83 26,71 31,06 23,80 31,99 31,28 32,55 32,44

A

N U SC RI PT

CC E

PT

ED

M

A

NANOG

R - ATGGGCTGCATCTGAGCGGC F - TCCAGCAAATGCAAGAACTTTC NM_001025344 R - TACATTTCATTCTCTGGTTCTGGAA

200 200 200

N/A

Figure legends Fig. 1. Presence of CXCR4 in testicular cells of pre-pubertal bull calves. A) Representative image of CXCR4 immunostaining of testicular tissue from pre-pubertal

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bulls. Inset depicts a 100% digital enlargement of a tubule containing a positive cell. B) Representative image of UCHL1 immunostaining of testicular tissue from pre-pubertal bulls. C) Representative image of a negative control for the immunostaining protocol. D) Representative dot-plot of isolated testicular cells analyzed by flow cytometry after incubation with CXCR4 antibody. Red dots represent unlabeled sample and blue dots

U

represent CXCR4 labeled sample. E) Graph displaying the percentage of CXCR4-

N

positive cells in the population of non-incubated cells, cells incubated only with

A

secondary antibody incubation and cells incubated with primary and secondary

M

antibodies. Images were obtained at a 400X magnification. Scale bar is equal to 100

D

µm.

TE

Fig. 2. Selection of CXCR4+ positive cells through MACS. A) Representative histogram of isolated testicular cells after MACS sorting using CXCR4 antibody. Red line and red

EP

area depict unsorted cells while blue line and blue area depict CXCR4+ cells as analyzed by flow cytometry. B) Graph displaying percentage of testicular CXCR4+ cells

CC

in unsorted cells incubated with CXCR4 antibody; and CXCR4- or CXCR4+ after MACS

A

sorting and flow cytometry analysis. C) Western blotting images from 4 different samples of CXCR4- and CXCR4+ cells using anti-CXCR4 antibody (top) or anti-alphatubulin antibody (bottom). Asterisk indicates significant statistical difference, n = 8. Fig. 3. Relative mRNA abundance analysis of CXCR4+ cells compared to CXCR4- (set to zero). Asterisk indicates significant statistical difference; n = 5.

Fig. 4. Flow cytometry analysis of cells sorted by CXCR4 MACS. A) Graph displaying percentage of testicular GFRA1+ cells in unsorted cells incubated with GFRA1 antibody; and CXCR4- or CXCR4+ cells incubated with GFRA1 antibody after flow cytometry

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analysis. B) Representative histogram of isolated testicular cells after MACS sorting using GFRA1 antibody. C) Graph displaying percentage of testicular THY1+ cells in unsorted cells incubated with THY1 antibody; and CXCR4- or CXCR4+ cells incubated with THY1 antibody after flow cytometry analysis. D) Representative histogram of isolated testicular cells after MACS sorting using GFRA1 antibody. Asterisk indicates

U

significant statistical difference; n = 5. In histograms, red line and red area depict

N

unsorted cells; green line and green area depict CXCR4- cells and blue line and blue

A

area depict CXCR4+ cells.

M

Fig. 5. Xenotransplantation of isolated testicular cells of bulls into testis of

D

immunocompromised mice depleted of germ cells. A) Representative image of an

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isolated seminiferous tubule presenting colonization by bovine CXCR4+ testicular cells as seen by red fluorescence. Arrowheads indicate chained cells. Asterisks indicate

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example of single cells or doublets that were not counted as colonies. Inset depicts a

CC

200% digital enhancement of a cell colony. B) Representative image of an isolated seminiferous tubule transplanted with CXCR4- testicular cells, in which no signs of

A

colonization were observed. Images were obtained at a 100X magnification. Scale bar is equal to 400µm. C) Graph displaying the observed average number of colonies per 100000 transplanted CXCR4+ or CXCR4- cells after xenotransplantation. The 95% confidence interval is depicted as error bars. Asterisk indicates significant statistical difference; n = 7.

A ED

PT

CC E A

M

D

N U SC RI P

A B

C

E

N U SC RI P B

CC E

PT

ED

M

A

A

A

C

CXCR4#3

CXCR4

α-Tubulin

#4

#5

CXCR4+ #7

#3

#4

#5

#7

A ED

PT

CC E A

M

N U SC RI P

A C

ED

PT

CC E A

M

N U SC RI P

A B

D

A ED

PT

CC E A

*

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*

N U SC RI P

A B

*

C

*