Primary Sertoli Cell Cultures From Adult Mice Have Different Properties Compared With Those Derived From 20-Day-Old Animals

Abstract

Cultures of Sertoli cells isolated from 20-day-old mice are widely used in research as substitutes for adult Sertoli cell cultures. This practice is based on the fact that Sertoli cells cease to proliferate and become mature in vivo by 16 to 20 days after birth. However, it is important to verify whether cultured Sertoli cells derived from 20-day-old mice do not proliferate ex vivo and whether they have the same properties as cultured adult Sertoli cells. Herein we described an isolation/culture method of Sertoli cells from 10-week-old adult mice with > 90% purity. Properties of these cultured adult Sertoli cells were then compared with those of cultured Sertoli cells derived from 20-day-old mice (also > 90% purity). By cell counting, bromo-2-deoxyuridine incorporation, and metaphase plate detection, we demonstrated that only adult Sertoli cells did not proliferate throughout 12 culture days. In contrast, Sertoli cells derived from 20-day-old mice still proliferated until Day 10 in culture. The morphology and profiles of intracellular lipidomics and spent medium proteomics of the 2 cultures were also different. Cultured adult Sertoli cells were larger in size and contained higher levels of triacylglycerols, cholesteryl esters, and seminolipid, and the proteins in their spent medium were mainly engaged in cellular metabolism. In contrast, proteins involved in cell division, including anti-Mullerian hormone, cell division cycle protein 42 (CDC42), and collagen isoforms, were at higher levels in Sertoli cell cultures derived from 20-day-old mice. Therefore, cultured Sertoli cells derived from 10-week-old mice, rather than those from 20-day-old animals, should be used for studies on properties of adult Sertoli cells.

Sertoli cells are fully differentiated somatic cells that occupy the whole width of the seminiferous epithelium in adult testes. They play a pivotal role in spermatogenesis by providing spatial and nutritional support to developing testicular germ cells (1, 2). Towards the basement membrane between adjacent Sertoli cells, there exist tight junctions and ectoplasmic specializations, which together form physical barriers to later meiotic and postmeiotic developing germ cells present in the adluminal compartments above these junctional complexes (3). Having specific transporters along the basolateral and apical membranes, Sertoli cells also act as permeability barriers regulating the movement of molecules in and out of the seminiferous tubule lumen. Thus, these 2 barriers restrict the entry of blood components into the adluminal compartments and the “non-self” developing germ cells sequestered in these compartments (3, 4) are protected from humoral attacks (3, 5, 6). In addition, a milieu advantageous for germ cell development is generated due to the property of these 2 barriers and the secretion of specific molecules by Sertoli cells, such as lactate, to be used as an energy substrate by spermatids (7, 8). Furthermore, Sertoli cells express a number of immunoregulatory factors, thus providing a local tolerogenic environment, which allows survival of germ cells that are not sequestered inside the adluminal compartments (9). All of these properties of Sertoli cells are the basis of blood-testis-barriers essential for completion of spermatogenesis (3, 6, 9).

The Sertoli cell lineage is already established in the fetus. Sertoli cells are central to fetal testis development. They are the first somatic cells to differentiate during the onset of fetal testis formation (10). Under support from follicle-stimulating hormone (FSH), fetal Sertoli cells undergo proliferation (11–14). Consequently, aggregates of Sertoli cells cluster encase germ cells to form testis cords (which become seminiferous tubules in adults), whereas peritubular myoid cells constitute the outer cell layer on the basal surface of Sertoli cells and fetal Leydig cells occupy the interstitial space between the testis cords. Through Sertoli cell signaling pathways, the differentiation of peritubular myoid cells and fetal Leydig cells is regulated. Concurrently, germ cells are prohibited from becoming mitotic and entering meiosis (10). Development of the Mullerian duct, which would result in female phenotype development, is also simultaneously inhibited by anti-Mullerian hormone (AMH) secreted by Sertoli cells (15–17).

Sertoli cell proliferation as induced by FSH continues during the neonatal period (13, 18). In humans and rhesus monkeys, another rise of Sertoli cell proliferation occurs before puberty (13). The increase in Sertoli cell numbers will eventually lead to a proportional increase in germ cells and testicular weight (19, 20). At puberty, Sertoli cells are mature, with the primary function of supporting spermatogenesis (as described above). The cessation of proliferation, formation of blood-testis barriers, and specific changes in protein expression are associated with Sertoli cell maturation (13,14).

Through various experimental approaches (detection of cells undergoing mitosis, ³H-thymidine in vivo incorporation/autoradiography, and cell counting through stereological analyses of testis sections), it is evident that Sertoli cells stop proliferating in vivo in rats/mice at 16 days of age and older (13, 21–29). These findings led to the consideration that Sertoli cells in rats/mice 16 to 20 days of age have started to develop properties of Sertoli cells of adult mice.

Primary cultures of Sertoli cells are needed for many studies that aim at evaluating their properties and roles in spermatogenesis. In 20-day-old rats/mice, Sertoli cells constitute ~20% of total cells in the seminiferous tubules, in contrast to only 3% to 5% of their distribution in the seminiferous tubules of adult rats/mice (28–31). Consequently, it is much easier to isolate Sertoli cells with > 90% purity from seminiferous tubules of rats/mice ~20 days of age and therefore, these primary cultures of Sertoli cells have long been used as surrogates of Sertoli cells from adult animals in in vitro studies (32–36). Although experts in the spermatogenesis field might realize that the primary culture of Sertoli cells isolated from 16- to 20-day-old mice likely differs from that derived from adult mice, to date there are no reports on the comparative properties of cultured Sertoli cells prepared from animals of these 2 ages. Therefore, the first objective of this report was to compare properties between cultures of Sertoli cells isolated from 20-day-old mice and those from 10-week-old mice (denoted as 20-day-SCs and 10-week-SCs, respectively).

In some instances where male infertility/subfertility stems from abnormality of Sertoli cells in adulthood, such as that observed in Arsa knockout mice (37), it is important that Sertoli cells can be isolated with high purity from adult animals for further studies. To date, only a limited number of reports describe protocols to isolate and culture Sertoli cells from adult rats/mice, and complete information on yield and purity has not yet been available (38–44). Thus, the second objective of our report is to describe this protocol with yield and purity results on Sertoli cell isolation from 10-week-old mice.

Materials and Methods

Animals

CD-1 male mice, 20 days old and 10 weeks old, obtained from Charles River laboratories (Senneville, QC, Canada) and kept in a temperature-controlled (22.5°C) room with a 12-hour light and 12-hour dark photoperiod, were fed ad libitum with Purina rodent chow and water. The use and handling of mice for all experiments adhered to the Canadian Council on Animal Care guidelines, with approval by the University of Ottawa Animal Care committee, which endorses the use of ARRIVE checklists and guidelines. Mice were sacrificed by cervical dislocation for testis collection.

Sertoli cell culture

As described previously (41, 46, 47), Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM-F12) supplemented with gentamycin (20 µg/mL), bacitracin (10 µg/mL) and fungizone (0.25 µg/mL) was used as isolation medium to prepare loose cells (Sertoli cells, testicular germ cells, and other co-isolated cells) from seminiferous tubules (prepared by denuding collected testes free of the tunica albuginea), whereas culture medium, which was isolation medium supplemented with insulin (10 μg/mL), transferrin (5.5 μg/mL), sodium selenite (0.0067 μg/mL), and epidermal growth factor (2.5 ng/mL), was used for culturing Sertoli cells. Loose cells were generated from seminiferous tubules through serial digestions with various enzymes (made in isolation medium), as previously described (39, 41, 48, 49), although we empirically determined the sequence of the sets of enzymes used, as described below, to give the highest yield of Sertoli cells. Isolation medium was used to prepare the 3 mixtures of the enzyme solutions (see below). For the last mixture (collagenase (1 mg/mL), hyaluronidase (2 mg/mL), DNase I (50 μg/mL), and soybean trypsin inhibitor (SBTI) (0.1 mg/mL), the pH of the solution was slightly adjusted to be 7.4 with 1 N NaOH. All steps in Sertoli cell isolation and culture were performed at 35°C.

For the protocol described below, 5 pairs of testes from 20-day-old mice and 3 to 5 pairs of testes from 10-week-old mice were used. Tissues/cells were processed in 50-mL Erlenmeyer flasks freshly coated with Millipore-Sigma Sigmacote solution, following manufacturer’s instruction. Sources of medium, reagents, enzymes, and specific plasticware used in Sertoli cell isolation are described in Reference (45).

All enzymatic treatments were performed in a shaking water bath, with continuous movement of 150 revolutions/minute. This was begun by treating (15 minutes) the testes denuded of tunica albuginea with the mixture of collagenase Type I (0.5 mg/mL) and DNase I (50 μg/mL) to disperse the seminiferous tubules, thus releasing interstitial cells into the surrounding. The supernatant was removed from the seminiferous tubules, which sedimented by gravity. The tubules were washed once in isolation medium and treated (10 minutes) with the mixture of 1 M glycine, 2 mM EDTA, and DNase I (50 μg/mL), made in isolation medium. This step and the previous step removed myoid cells and Leydig cells from the tubules (48–50). Following washing in isolation medium, the tubules were digested with the mixture of trypsin (0.5 mg/mL) and DNase I (50 μg/mL) for 15 minutes. At the end of the treatment period, SBTI (0.5 mg/mL) was added into the suspension. The tubules, which were fragmented into small pieces, were then washed twice in isolation medium by centrifugation (800g, 3 minutes). These tubule fragments were further digested (30 minutes) with the mixture of collagenase (1 mg/mL), hyaluronidase (2 mg/mL), and DNase I (50 μg/mL) in the presence of SBTI (0.1 mg/mL) to generate loose cells (> 90%). At this point, elongated spermatids released from Sertoli cell recesses and testicular sperm from the lumen were also fragmented into heads and tails. All loose cells, heads and tails of elongated spermatids and testicular sperm, and residual tubule particulates were then washed 3 times by centrifugation (800g, 3 minutes) in isolation medium. Using a 100 μm cell strainer, the residual tubule particulates in the suspension were filtered away from the single cell suspension, which was then diluted 3× in isolation medium and subjected to centrifugation at 50g for 5 minutes (43). Sertoli cells and testicular germ cells of higher density were sedimented together as a pellet, whereas lighter testicular germ cells and the majority of heads and tails of testicular sperm and elongated spermatids remained in the supernatant, which was then discarded. Pelleted cells resuspended in culture medium were counted, and the cell suspension was plated onto a Sarstedt 10-cm polystyrene tissue culture dish in 10 mL of culture medium. The process starting from testis decapsulation to this point took approximately 4 hours.

For isolation from 20-day-old mice, 7 million to 10 million cells (mixture of Sertoli cells, testicular germ cells, and a small number of co-isolated myoid cells) were used for plating per dish, whereas for 10-week-old mice, the corresponding plating number was approximately 30 million to 38 million cells. A higher number of cells were used for plating in the latter because the percentage of Sertoli cells in the seminiferous tubules is significantly lower in adult animals.

Sertoli cells, testicular germ cells, and a small number of myoid cells in the dish were cultured at 35°C under 5% CO₂. Adherent Sertoli cells and myoid cells were attached to the dish within 24 hours. On culture Day 2 (Day 1 = the day that the cells were plated onto the dish), the dish was gently swirled to float testicular germ cells into the medium, which was then gently removed using a transfer pipet. Fresh culture medium was then added into the culture. This step was repeated daily until Day 3 for 20-day-SCs and until Day 4 for 10-week-SCs. Medium was subsequently replaced every 2 days until Day 8. Afterwards, when the culture showed a sign of acidity as observed by the color change of the pH indicator in spent medium, half of the spent medium was removed and replaced by the same volume of fresh medium. Morphology of cells in the plate was monitored daily by bright-field microscopy under a Nikon TMS inverted microscope using a 10× objective with a Celestron HD Digital Microscope Imager (Torrance, California, containing a 30× magnifier lens) attached to an eyepiece. Attention was paid to the presence of Sertoli cells, scored by their possession of tripartite nucleoli and their cytoplasmic spread to be next to each other on the plate. In addition, we closely monitored the decreasing numbers of round testicular germ cells, which were removed during medium replacement.

When the Sertoli cells adhered well to the plate, with minimal numbers of myoid cells in the culture dish and no round testicular germ cells remaining, the Sertoli cells were detached from the culture plate by treatment with 4 mL of Accutase Cell Detachment Solution (5 minutes, 35°C, 5% CO₂). Culture medium (4 mL) was then added to the detached cell suspension. Harvested Sertoli cells were washed twice in medium by centrifugation (500g, 5 minutes), and an aliquot was counted under a microscope using a hemocytometer. Collected cells were used in some experiments for biochemical work.

In another set of experiments, the mixture of Sertoli cells and testicular germ cells were seeded into an ibidi high-walled μ-Dish 35-mm plate (ibidi USA, Inc., Fitchburg, Wisconsin), which contains a polymer coverslip for cell culture in the dish bottom. The number of the mixed Sertoli cells, testicular germ cells, and a small number of co-isolated myoid cells (prepared as described above) used for seeding per ibidi plate was 0.5 million to 1 million for cells obtained from the 20-day-old mice and was 8 million for cells from the 10-week-old males. Culture conditions in ibidi plates were the same as those described for the 10-cm tissue culture dishes. Cultures in an ibidi dish were most useful for detection of contaminated heads of testicular sperm and elongated spermatids, which could adventitiously bind to the surface of Sertoli cells. This was done by fluorescent staining of the nuclei (including heads of sperm and elongated spermatids) with Hoechst 33258 (see Immunofluorescence section below).

Cellular dimension measurement

Lengths and widths of 20-day-SCs and 10-week-SCs cultured on 10-cm plates until Day 5 and Day 8, respectively, when the cells were at their largest, were measured using National Institutes of Health (NIH) ImageJ software following instructions on the NIH website (https://imagej.nih.gov/ij/docs/guide/). Measurement was performed on 100 Sertoli cells in total for each sample.

Immunofluorescence

20-day-SCs and 10-week-SCs cultured in ibidi plates were used for all immunofluorescence experiments. All incubation steps were done at room temperature (RT) unless indicated otherwise. Sources, Research Resource Identifier (RRID) and dilution of each antibody are shown in Table 1.

Table 1.

Identities of Antibodies Used in Our Experiments¹

Antibody	RRID No.1	Host Species	Vendor	Catalog No.	Working Dilution
					IF2	IB3
Wilms tumor protein 1	AB_2784516 (51)	Rabbit	Abcam,	ab180840	1:100	−
α smooth muscle actin	AB_476701 (52)	Mouse	Sigma-Aldrich	A2547	1:500	−
COUP-TF II/NR2F2	AB_2155627 (53)	Mouse	R&D Systems	PP-H7147-00	1:100	−
Clusterin / APOJ	AB_1556289 (54)	Goat	Novus Biologicals	NBP1-06027	1:50	−
Claudin-11 (H-107)	AB_639330 (55)	Rabbit	Santa Cruz Biotechnology	sc-25711	1:50	1:200
ZO-1	AB_2533456 (56)	Rabbit	Invitrogen	40–2200	1:100	1:500
β-Tubulin	AB_609915 (57)	Mouse	Sigma-Aldrich	T5201	1:500	1:1000
BrdU-Alexa Fluor 488	AB_2536434 (58)	Mouse	Invitrogen	B35130	1:100
AMH	AB_2544438 (59)	Rabbit	Invitrogen	PA5-26938	−	1:1000
SHBG (M-207)	AB_2187753 (60)	Rabbit	Santa Cruz Biotechnology	sc-32891	−	1:200
Glutathione Peroxidase 3/ GPX3	AB_2112276 (61)	Goat	R&D Systems	AF4199	−	1:400
Prosaposin (PSAP)	AB_2792974 (62)	Rabbit	Dr. Carlos Morales, Mcgill University, Montreal		−	1:5000
CDC42	AB_1310067 (63)	Rabbit	Abcam	ab64533	−	1:1000
p27Kip1	AB_2544664 (64)	Rabbit	Thermo Fisher Scientific	PA5-27188	−	1:1000
Goat IgG-Alexa Fluor 594	AB_2534105 (65)	Donkey	Thermo Fisher Scientific	A-11058	1:200	−
Rabbit IgG-Alexa Fluor 594	AB_2534079 (66)	Goat	Thermo Fisher Scientific	A-11012	1:300	−
Mouse IgG-Alexa Fluor 488	AB_2534069 (67)	Goat	Thermo Fisher Scientific	A-11001	1:500 (SMA) 1:200 (COUP-TFII)	−
Rabbit IgG-Alexa Fluor 488	AB_2576217 (68)	Goat	Thermo Fisher Scientific	A-11034	1:200 (ZO1) 1:400 (Claudin11)	−
Goat IgG-HRP	AB_11125144 (69)	Rabbit	Bio-Rad Laboratories	1721034	−	1:5000 (CLU) 1:2000 (GPX3)
Rabbit IgG-HRP	AB_11125142 (70)	Goat	Bio-Rad Laboratories	1706515	−	1:500 (Caludin-11) 1:1000 (ZO-1) 1:5000 (other rabbit primary antibodies)
Mouse IgG-HRP	AB_11125547 (71)	Goat	Bio-Rad Laboratories	1706516	−	1:2000
Normal Rabbit IgG	AB_737196 (72)	Rabbit	Santa Cruz Biotechnology	sc-3888	1:20	−
Normal Mouse IgG	AB_737182 (73)	Mouse	Santa Cruz Biotechnology	sc-2025	1:20	−

¹RRID No. was retrieved from http://antibodyregistry.org.

²IF = immunofluorescence

³IB = immunoblotting

Purity assessment

Wilms tumor protein 1 (WT1) protein was used as a specific marker of Sertoli cells (74), whereas α-SMA and COUP-TFII served as markers for myoid (peritubular) cells and Leydig cells, respectively (75, 76). Since Leydig cells and myoid cells were either absent or present at very low levels in Sertoli cell cultures, immunofluorescence protocols for COUP-TFII and α-SMA, respectively, were first tested with Leydig cells released from seminiferous tubules after treatment with the first set of enzymes (collagenase + DNase I) and with myoid cells dissociated from the basement membrane of seminiferous tubules after treatment with 1 M glycine + DNase I (as described above). The purity of Sertoli cells on culture Days 4 to 6 for the 20-day-SCs and culture Day 7 for the 10-week-SCs was then assessed by triple immunofluorescence with antibodies against the 3 cell markers. Briefly, the culture was fixed in cold methanol prechilled at −20°C for 10 minutes. To block nonspecific interaction, the culture was treated (1 hour) with phosphate-buffered saline (PBS) containing 4% bovine serum albumin (BSA) prior to incubation with all primary and secondary antibodies, and the culture was washed in PBS after each treatment step. Immunofluorescence was started with incubation of the culture with anti-WT1 (1 hour) followed by its secondary antibody (Alexa-Fluor-594 conjugated goat anti-rabbit IgG, 1 hour). Immunofluorescence proceeded with anti-α-SMA (1 hour) and its secondary antibody (Alexa-Fluor-488 conjugated goat anti-mouse IgG, 1 hour). The culture was then treated with 0.1% Triton X-100 (10 minutes) prior to the immunofluorescence steps with anti-COUP-TFII (overnight) and its secondary antibody (Alexa-Fluor-488 conjugated goat anti-mouse IgG, 1 hour). Although the fluorescent secondary antibody was the same for anti-α-SMA and anti-COUP-TFII, the 2 antigens were localized at different sites: the cytoplasm for α-SMA and the nucleus for COUP-TFII. And therefore, these 2 antigens could be differentially identified. After all immunofluorescence steps, the culture was incubated (5 minutes) with Hoechst 33258 (5 µg/mL, Molecular probes: Cat No. H-21491), then washed, topped with 1 mL of PBS, and viewed for immunofluorescence signals under a Zeiss Axio epifluorescence microscope. Fluorescent images were taken using the rhodamine filter for WT1 detection, the fluorescein filter for α-SMA and COUP-TFII, and the Hoechst filter for Hoechst staining. Images from the 3 filters were merged for presentation.

A negative control of immunofluorescence for WT1 and α-SMA/COUP-TFII was the parallel Sertoli cell culture, which was fixed and incubated with normal rabbit IgG (in place of rabbit anti-WT1) and normal mouse IgG (in place of mouse anti-α-SMA and mouse anti-COUP-TFII) in the same consecutive manner (including blocking, washing, Triton X-100, Hoechst staining, and image viewing/recording steps), as described above for the immunofluorescence of the 3 antigens.

Detection of Sertoli cell proteins (claudin-11 and ZO-1).

On culture Day 4 to Day 6 for 20-day-SCs and on culture Day 7 for 10-week-SCs, the cells were fixed in 4% paraformaldehyde made in PBS (PFA/PBS) (10 minutes, RT) followed by plasma membrane permeabilization by treatment (10 minutes) with 0.1% Triton X-100 in PBS. Blocking of nonspecific binding and other procedural steps were the same as that described above.

Detection of metaphase plates in dividing Sertoli cells

This was through immunofluorescence staining of β-tubulin. The 20-day-SCs cultured till Day 4 were used for this detection. Cell fixation and permeabilization steps and other technical steps were similar to those described for the detection of Sertoli cell proteins above, although the Triton X-100 treatment step was 30 minutes and the primary antibody incubation was at 4°C overnight.

Lipid droplet staining

Oil Red O was used to detect lipid droplets. Oil Red O stock solution (0.5% in isopropyl alcohol) was diluted with PBS to the working concentration of 0.3% and filtered through a Whatman No. 1 filter paper. The 20-day-SCs cultured on ibidi plates until Day 5 or 6 and 10-week-SCs cultured on ibidi plates until Day 7 were fixed (RT, 30 minutes) in 4% PFA/PBS and then incubated (RT, 15 minutes) with Oil Red O. To remove unincorporated Oil Red O, the cells were washed with 70% ethanol and further washed several times with PBS. Finally, they were counterstained with Harris hematoxylin for 30 seconds. Following washing in PBS, the cells were viewed under a Zeiss Axioskope microscope. The numbers of Sertoli cells containing lipid droplets were counted in 3 replicate cultures of 20-day-SCs and 10-week-SCs, including 19 areas of 1028 cells and 25 areas of 1065 cells, respectively.

Detection of DNA replication by 5-bromo-2-deoxyuridine incorporation

20-day-SCs and 10-week-SCs cultured on ibidi dishes for various days were used for this assessment. They were incubated (35°C, 5% CO₂) for 48 h with 10 μM Molecular Probes 5-bromo-2-deoxyuridine (BrdU) (Thermo Fisher, Nepean, ON, Canada) made in culture medium. After successive washing with PBS to remove unincorporated BrdU, they were fixed with 4% PFA/PBS (15 minutes, RT) and subsequently permeabilized with 0.5% Triton X-100 (30 minutes, RT). The fixed and permeabilized cells were washed 3 times with PBS and treated (4°C, 15 minutes) with 1 N HCl and then with 2 N HCl (RT, 15 minutes). At this step, histones were solubilized and DNA denatured, thus exposing incorporated BrdU. The cells were then neutralized by rinsing and incubation (10–20 minutes) in PBS and washed 3 times (2 minutes each) with PBS containing 0.5% Triton X-100. Subsequently, the cells were blocked for nonspecific binding (RT, 15 minutes) with 4% BSA in PBS prior to incubation (RT, overnight) with Alexa Fluor 488-conjugated anti-BrdU. After successive washing in PBS, the nuclei were fluorescently stained with Hoechst 33258 and viewed under the Zeiss Axio epifluorescence microscope. Experiments were repeated 3 times for all culture periods of 20-day-SCs and 10-day-SCs and a sample of at least 125 cells on each experimental day was assessed for percentage of BrdU-positive cells.

Treatment of Sertoli cell cultures with FSH

20-day-SCs and 10-week-SCs cultured on ibidi plates until Day 18 and Day 9, respectively, were treated (35°C, 48 h) with 1 IU/mL of recombinant human FSH (rhFSH) (National Hormone & Peptide Program, Cat No:AFP8468A) (Torrance, California). They were then analyzed for DNA replication activity by BrdU incorporation as described above. Parallel cultures that were not treated with rhFSH served as negative controls.

Lipidomic analyses

Similar numbers of 20-day-SCs and 10-week-SCs cultured for 6 and 7 days, respectively, on 10-cm tissue culture dishes and harvested by Accutase treatment were subjected to lipid extraction by a modified Bligh-Dyer method (77). Major lipid classes in isolated Sertoli cells were identified by different modes of tandem mass spectrometry using an Agilent 6460 triple quadrupole mass spectrometer (MS) (Agilent Technologies, Santa Clara, California) as previously described (78). Briefly, lipids extracted from 0.04 million cells each from 20-day-SCs and 10-week-SCs were used for each MS/MS analysis (10-μl injection). Precursor ion scanning in the positive ion mode for parents of m/z 184, 364.9, and 264.1 was used to identify phosphatidylcholines (PCs)/sphingomyelins (SMs), cholesteryl esters (CEs) and ceramides (Cers), respectively. Precursor ion scanning in the negative ion mode was performed for identification of sulfolipids (parents of m/z 97) and phosphatidylinositols (PIs) (parents of m/z 241). Neutral loss scanning in the positive ion mode was used to identify phosphatidylethanolamines (PEs) (loss of m/z 141) and the major species of triacylglycerols (TAGs; losses of 273 and 299, which correspond to C16:0 and C18:1 acyl chains, respectively). The major signals from each scanning mode were tentatively identified on the basis of mass concordance between the calculated and measured masses and were selected for further quantitative analyses by multiple reaction monitoring (MRM) using the transitions described in Reference (45). For each MRM analysis, the extracted lipids corresponding to 0.008 million Sertoli cells were used per injection (10 μL). The peak area of each parent to fragment ion transition obtained from the MRM analyses was measured using instrument manufacturer-supplied software (Agilent Mass Hunter version B.05.00). The lipidomic analysis experiments were performed twice. For each experiment, all lipid analyses were done in triplicates.

Quantification and analyses of proteins present in spent Sertoli cell medium

Spent medium was collected from cultures of Sertoli cells. The collection was from culture Day 4 to Day 6 for 20-day-SCs, and from Day 6 to Day 8 for 10-week-SCs. Five and 4 sets of medium from different culture experiments were collected from 20-day-SCs and 10-week-SCs, respectively. The medium was extensively dialyzed against 1 mM Tris-HCl, pH 7.4 and lyophilized. The lyophilized materials were then solubilized in a small volume of water and a small aliquot was used for protein quantification by the Bradford assay. The solution was then re-lyophilized and used for SWATH MS analyses as well as immunoblotting.

Sequential window activation of all theoretical masses (SWATH) MS analysis

Protein digestion was performed as described previously (79). Peptide (5 µg) were dissolved in loading buffer (2% acetonitrile, 0.1% formic acid) and injected using a Agilent 1200 high-pressure liquid chromatography (HPLC) instrument (Agilent Technologies) equipped with a trapping column (75 μm × 15 cm ChromXP, C18, 3 μm, 120 Å) and C18 RP column (ChromXP C18, 3 µm 120 Å 15 cm). Peptides were separated over a 70-minute gradient from 5% to 98% solvent before ionization (DuoSpray ion source, AB Sciex, Framingham, MA, USA). For the generation of the spectral library, the Triple ToF q6600 mass spectrometer (AB Sciex) was operated in data-dependent analysis (DDA), whereby the top 20 most abundant ions were selected for presentation in the positive ion, with 20-second dynamic exclusion. Rolling collision energy was applied with a spread of 5 eV that was used for fragmentation. One sample randomly selected from each group of Sertoli cell samples (n = 5 for 20-day-SCs and n = 4 for 10-week-SCs) was subjected to DDA analysis. The resulting files were used to generate a peptide library by ProteinPilot v5.0.1 (AB Sciex) with the following search parameters: trypsin as the cleavage enzyme with 2 missed cleavages and carbamidomethylation as a fixed modification of cysteines. Within the ProteinPilot instrument setting option, MS tolerance was preset to 0.05 Da and MS/MS tolerance to 0.1 Da. The search was carried out in “thorough ID” mode with a detected protein threshold of 1% plus false discovery rate (FDR) analysis, matching to the Mus musculus SwissProt database downloaded May 2017 (25043 proteins).

For SWATH-MS data acquisition, the same mass spectrometer and LC-MS/MS setup was used essentially as described above, but operated in SWATH mode. SWATH experiments were conducted with a variable Q1 window width with 1 Da overlap covering the m/z range from 400 to 1250 in high-sensitivity product ion scan mode. The overall cycle time was 2.5 seconds.

Data processing.

The spectral library generated from the DDA files was imported into PeakView 2.0 (AB Sciex). The retention times for the SWATH file were aligned by a linear equation calculated using shared peptides. To match the SWATH data to the library file, thresholds were set at a 99% peptide confidence, 1% FDR and shared peptides were excluded. Prior to spectral library matching, the XIC width was set at 20 ppm. After matching to library files, peak areas were exported into MarkerView 1.2 (AB Sciex), then exported as a tab-separated text file. Using the freely available software, Perseus (http://www.perseus-framework.org), the raw ion count values were log₂ transformed, and median normalized. Significant changes were determined by the Student’s t-test with a P value of < 0.05, with further correction by Benjamani-Hochberg analyses. Only proteins with a fold change of >2 (log₂ fold change > 1) were included in our analyses. In our setup, the log₂ fold change value was positive, when the ion counts were higher in the 10-week-old group. The reverse (negative log₂ fold change value) was observed with higher ion counts in the 20-day-old group.

Isolation of proteins from Sertoli cells

Approximately 2 × 10⁶ Sertoli cells on culture Day 6 of 20-day SCs and 400 000 Sertoli cells on culture Day 7 of 10-week-SCs, harvested as described above, were treated (90°C, 5 minutes) with 50 µl of 2% sodium dodecyl sulfate (SDS) in 0.0625 M Tris-HCl, pH 6.8. The treated cells were then centrifuged (14,000g, 5 minutes) and proteins in the supernatant were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific) and subjected to immunoblotting.

Immunoblotting

Proteins in the Sertoli cell spent medium and those extracted from Sertoli cells were adjusted to be in Laemmli SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (80) and reduced with 100 mM dithiothreitol (DTT). Solubilized proteins were subjected to Laemmli SDS-PAGE on an 8% or 10% polyacrylamide gel. Following electrophoresis, the proteins were electro-transferred (100 V, 1 hour) (81) onto a nitrocellulose membrane (Bio-Rad Laboratories) for immunoblotting. The membrane was blocked (RT, 1 hour) for nonspecific binding in 5% nonfat skim milk in TBST (10 mM Tris, pH 7.9, 150 mM NaCl, 0.1% Tween 20). Incubation of proteins with the primary antibodies against ZO-1, claudin-11, prosaposin (PSAP), androgen-binding protein (ABP) (also known as sex hormone–binding globulin [SHBG]), anti-Mullerian hormone (AMH), glutathione peroxidase 3 (GPX3), CDC42, and p27^Kip1, all diluted in TBST-5% skim milk with dilution factors described in Table 1, was at 4°C for 12 hours, whereas incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in TBST-5% skim milk (see Table 1) was at RT for 1 hour. In between different incubation steps, the membrane was washed successively with TBST (RT, 10 minutes for each wash). Reactivity of antigen with antibody was revealed by enhanced chemiluminescence (ECL) on a GE Healthcare Amersham Hyperfilm (Thermo Fisher Scientific) using a Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) for most antigen-antibody interactions. For antigens with weak interaction with their antibody (i.e., AMH, claudin-11, and ZO-1), a SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific) was used in place of the Pierce ECL substrate.

Statistical analyses

Significant differences between values obtained from 20-day-SCs and 10-week-SCs were analyzed by the Student’s t test.

Results

Purity and yield of Sertoli cells isolated from 20-day-old and 10-week-old mice

Following all the steps of enzymatic treatment of seminiferous tubules and then filtration of the obtained cell suspension through a 100-μm cell strainer, only loose cells (a mixture of Sertoli cells, testicular germ cells, and a very small number of co-isolated myoid cells) were present in the filtrate. For Sertoli cell isolation from 10-week-old mice, heads and tails of elongated spermatids and testicular sperm were also present (data not shown). This indicated that the enzymes had disrupted junctional complexes, which held Sertoli cells together in seminiferous tubules of animals of both ages, as well as the apical ectoplasmic specializations, which captured the elongated spermatid heads in the Sertoli cell recesses in seminiferous tubules of 10-week-old mice. In addition, the enzymes dissociated elongated spermatids and testicular sperm into heads and tails, the majority of which, together with round spermatids, remained in the supernatant upon centrifugation of the cell mixture at 50g. The suspension of pelleted cells was composed mainly of Sertoli cells and heavy testicular germ cells (e.g., primary spermatocytes) and a small number of myoid cells. Once plated into a culture dish, only Sertoli cells and myoid cells attached to the substratum within 24 hours. By Day 4 and Day 6 of 20-day-SCs and 10-week-SCs, respectively, more than 90% of the adherent cells possessed condensed nucleoli, frequently appearing as a tripartite structure (45), characteristic of Sertoli cells. Round testicular germ cells were completely removed from the culture during medium changes (45). For 10-week-SCs, the heads of testicular sperm and/or elongated spermatids were present at 3.7%, as shown by superimposed differential interference contrast and Hoechst stained images (45).

The numbers of the mixture of Sertoli cells, round testicular germ cells, and co-isolated myoid cells for plating into a culture dish were empirically determined for the outcomes of high purity and an optimum yield of Sertoli cells, as well as the ability of Sertoli cells to spread out to touch each other to form tight junctions. Sertoli cells in 20-day-old mice constitute ~20% of total seminiferous tubule cells (29, 31, 82), whereas the percentage of Sertoli cells in the seminiferous tubules of 10-week-old mice is only 3% to 5% (28, 29, 31). Therefore, the plating number of mixed Sertoli cells, testicular germ cells and myoid cells obtained from 10-week-old mice for a 10-cm dish and a 35-mm ibidi dish was ~ 4× and 8×, respectively, higher than the plating number of the corresponding mixed cells prepared from 20-day-old animals. Based on the fact that WT1 was a specific marker for Sertoli cells (74), the purity of Sertoli cells (WT1-positive as shown by immunofluorescence) (Fig. 1A) among all adherent cells was 99.8% ± 0.1% for 20-day-SCs (n = 3 experiments, with total 1382 cells counted) and 97.3% ± 2.5% for 10-week-SCs (n = 4, with total 2819 cells counted). Although % purity was only slightly lower in 10-week-SCs, it was significantly lower from that of 20-day-SCs (P < 0.05) (Fig. 1B). A very small percentage of myoid cells (α-SMA positive) were co-isolated adherent cells present in the cultures derived from both ages. In contrast, Leydig cells with COUP-TFII as their marker were not present in the cultures (Fig. 1A).

Figure 1.

Purity and yield of Sertoli cells isolated from 20-day-old and 10-week-old mice. (A) Triple immunofluorescence for WT1 (a marker of Sertoli cells), α-SMA (a marker of myoid cells) and COUP-TFII (a marker of Leydig cells) of 20-day-SCs and 10-week-SCs. Merged fluorescent images obtained under the rhodamine filter (for WT1 detection), fluorescein filter (for α-SMA and COUP-TFII detection) and Hoechst filter were shown (see Materials and Methods for details). Negative controls were 20-day-SCs and 10-week-SCs that were processed in parallel but were incubated with normal rabbit IgG and normal mouse IgG instead of the primary antibodies and the negative control for 10-week-SCs (45). (B) Percentage of cells showing WT1 signal in 20-day-SCs and 10-week-SCs. Data are expressed as mean ± SD from 3 and 4 replicate experiments for 20-day-SCs and 10-week-SCs. (C) Yield of Sertoli cells isolated from 20-day-old and 10-week-old mice. Data are shown as mean ± SDs of million cells/mouse from 4 and 5 replicate experiments of 20-day-SCs and 10-week-SCs, respectively.

After 7 days in culture, the yield of Sertoli cells was ~2.2 million per mouse in 20-day-SCs, whereas the corresponding yield was only 0.5 million per mouse in 10-week-SCs (Fig. 1C). Both 20-day-SCs and 10-week-SCs, by culture Day 4–6 and Day 7, respectively, had properties typical of Sertoli cells existing in testes in situ (83). The cells contacted each other and contained the tight junction proteins claudin-11 and ZO-1 (Fig. 2). These cultured Sertoli cells also contained clusterin (data not shown), known to be expressed by Sertoli cells in vivo (83,84), as well as tripartite nuclei (45). It is noted, however, that levels of claudin-11 and ZO-1 were much lower in 20-day-SCs, as compared with 10-week-SCs, as shown by both immunofluorescence and immunoblotting (Fig. 2).

Figure 2.

Presence of the tight junction proteins claudin-11 and ZO-1 in 20-day-SCs and 10-week-SCs. 20-day-SCs on culture Day 4–6 and 10-week-SCs on culture Day 7 were subjected to immunofluorescence (A) and immunoblotting (B) for claudin-11 and ZO-1. For immunofluorescence experiments, negative controls were the cultures that were processed in parallel except that normal rabbit IgG was used in place of the primary antibody. Results shown are representative of 3 replicate experiments. For immunoblotting, the same number of cells of 20-day-SCs and 10-week-SCs (60 000 for claudin-11 and 30 000 for ZO-1) were used for protein extraction and gel loading. The densities of claudin-11 band and ZO-1 band were both 2.5× higher in 10-week-SCs compared with 20-week-SCs. Results shown were representation of 2 replicate experiments.

Morphology of cultured Sertoli cells isolated from 20-day-old and 10-week-old mice

Both 20-day-SCs and 10-week-SCs attached to the substratum of a 10-cm culture dish within 24 h. For 20-day-SCs, the Sertoli cells were fully spread, contacting one another, and appeared cuboidal by Day 3 in culture. In 10-week-SCs, the cell spreading was more prominent on Day 5 or Day 6, with irregular patterns, leading to formation of cellular processes (Fig. 3). The length and width dimensions of 20-day-SCs and 10-week-SCs, when their sizes were largest in culture, were significantly different from each other: 39.5 ± 8.0 μm (width) and 57.8 ± 8.5 μm (length) for 20-day-SCs versus 61.0 ± 13.3 μm (width) and 89.5 ± 13.4 μm for 10-week-SCs (45). Interestingly, on culture Day 5 and later on, 20-day-SCs became progressively smaller in size and their numbers appeared to increase. On Day 8, a number of these Sertoli cells piled up above the substratum-attached cell layer (Fig. 3). This observation implied that 20-day-SCs were dividing during the culture. Similar morphological changes were observed when the culture was done in a 35-mm ibidi dish, although the cell spreading in both 20-day-SCs and 10-week-SCs and the increase in cell numbers for 20-day-SCs were about 15 to 24 hours slower than those occurring in a 10-cm culture dish.

Figure 3.

Morphological changes of Sertoli cells in culture: comparison between 20-day-SC and 10-week-SC cultures. The mixture of loose Sertoli cells and testicular germ cells were seeded on Day 1 onto a culture plate and observed for morphological changes as a function of culture time.

Lipidomic analyses

In addition to differences in size as described above, 20-day-SCs and 10-week-SCs showed a drastic difference in possession of lipid droplets (stained with Oil Red O). Among more than 1000 cells evaluated in 3 replicate experiments, only 5.5% ± 5.7% of 20-day-SCs contained lipid droplets, in contrast to 87.7% ± 7.2% in 10-week-SCs (P < 0.0001) (Fig. 4). Since CEs and TAGs are, in general, main components of lipid droplets (85), including those isolated from testes (86), quantitative MS-based lipidomic analyses were performed to determine amounts of these 2 neutral lipids. Using the same cell numbers of 20-day-SCs and 10-week-SCs for these MS analyses, the data obtained corroborated the lipid droplet staining results. Levels of various molecular species of the 2 major neutral lipids, TAGs and CEs, were significantly higher in 10-week-SCs (Fig. 5). Since Sertoli cells are actively engaged in phagocytosing apoptotic testicular germ cells and residual bodies of elongated spermatids (3, 87, 88), both of which contain sulfogalactosyl glycerolipid (SGG) (5), SGG was also quantified by MS-MRM in both 20-day-SCs and 10-week-SCs. As expected, Sertoli cells from 10-week-old mice, which had been through a number of spermatogenesis rounds, contained almost double the amount of SGG, as compared with 20-day-SCs (Fig. 5). Levels of the 2 main molecular species of Cers and PIs) were also higher in 10-week-SCs (45). Two molecular species of SMs also showed higher levels in 10-week-SCs and 1 molecular species showed a lower amount, relative to 20-day-SCs. In contrast, levels of various molecular species of PCs and PEs in 20-day-SCs and 10-week-SCs did not have significant differences (45).

Figure 4.

Lipid droplets in cultured Sertoli cells. 20-day-SCs and 10-week-SCs, cultured to Day 5–6 and Day 7, respectively, were stained for lipid droplets with Oil Red O. Quantification of % Sertoli cells having lipid droplets was performed in 19 and 25 microscopic fields over 1000 cells from 3 replicate experiments of 20-day-SCs and 10-week-SCs, respectively, and the data were expressed as mean ± SD.

Figure 5.

Relative abundance of major species of triacylglycerols (TAGs) and cholesteryl esters (CEs), and SGG (C16:0/16:0) in 20-day-SCs and 10-week-SCs. Lipids extracted from an equal cell number of the 2 Sertoli cell cultures were subjected to different modes of ESI-MS/MS analyses to profile different classes of lipids. Major species of TAG and CE identified, and SGG (C16:0/C16:0), the only sulfolipid identified, were subjected to MRM analyses to compare their relative abundance in Sertoli cells isolated from mice of the 2 ages. Data (peak areas) are expressed as mean ± SD averaged from 3 replicate samples. Asterisks indicate statistically significant differences between the samples from 20-day-old and 10-week-old mice (*P < 0.05; **P < 0.001). The numbers in parenthesis were m/z values of the lipid ions. Their potential identities are listed in Reference (45). Data presented are representative of 2 replicate experiments.

Proliferation of Sertoli cells isolated from 20-day-old mice in culture

The cell number of 20-day-SCs increased almost twofold from Day 3 to Day 8 in culture, indicating that these cultured Sertoli cells still proliferated (Fig. 6A). The proliferation activity of these Sertoli cells was further confirmed by the presence of metaphase plates in a small population of cells (0.07%, 0.03%, and 0.02% of total cells in the 3 replicate experiments) during this culture period (Fig. 6B). However, at later days in culture, Day 12 and Day 15, these Sertoli cells decreased in number (14.3% and 21.7% decrease, compared with the maximum cell number on Day 8). This decrease could be from the sloughing of Sertoli cells, which had piled onto the pre-existing Sertoli cell monolayers (as shown in Fig. 3), off the plate during medium changes.

Figure 6.

20-day-SCs but not 10-week-SCs proliferated in culture as shown by cell counting (A) and the presence of metaphase plates in 20-day-SCs (B). Cell counting started on Day 3 and Day 4 for 20-day-SCs and 10-week-SCs, respectively, since the majority of cells attached to the plate on these culture days were Sertoli cells. Data are shown as mean ± SD of 3 replicate experiments.

The ability of 20-day-SCs to proliferate in culture was further shown by nuclear incorporation of BrdU (Fig. 7A). BrdU incorporation was present in 82.8% ± 3.4%, 54.7% ± 3.5% and 19.4% ± 4.4% of total Sertoli cells during culture Day 3 to Day 4, Day 5 to Day 6, and Day 7 to Day 8, respectively, in 20-day-SCs. However, during culture Day 9 to Day 10 of 20-day-SCs, the number of Sertoli cells showing incorporated BrdU was much decreased. Only 22.3% ± 3.8% of the Sertoli cells in the middle of the plate (totaling to ~2.2% of all cells) showed BrdU incorporation, whereas the remaining cells in the plate periphery did not incorporate BrdU. By Days 15 and 16, there was no BrdU incorporation in the whole culture plate of 20-day-SCs (Fig. 7A).

Figure 7.

Only cultured Sertoli cells isolated from 20-day-old mice incorporated BrdU. Cultures of Sertoli cells were incubated for 48 hours with BrdU during the culture Day 3–4, Day 5–6, Day 7–8, Day 9–10, and Day 15–16 for 20-day-SCs (A) and during the culture Day 4–5 and Day 10–11 for 10-week-SCs (B). Percentages of cells that incorporated BrdU (with a green fluorescent signal) were indicated at the top left corner of each panel. Data are expressed as mean ± SD of means from 3 replicate experiments and the numbers of cells analyzed were 450 or more for each culture period. Note that on culture Day 9–10 of 20-day-SCs, the cells that incorporated BrdU were mainly localized in the middle of the plate. Cells in the plate periphery did not show any fluorescent signal. None of 10-week-SCs incorporated BrdU on either Day 4–5 or Day 10–11 in culture.

In contrast, 10-week-SCs did not increase in number when assessed during culture Day 4 to Day 12 (Fig. 6A). Corroborating this result, none of these Sertoli cells incorporated BrdU during culture Day 4 to Day 5 and Day 10 to Day 11 (Fig. 7B).

Effects of rhFSH on Sertoli cell proliferation

20-day-SCs showed no BrdU incorporation on Day 18 in culture. However, when these Sertoli cells were treated (35°C, 48 hours) with rhFSH (1 IU/mL), 68% of them were able to incorporate BrdU (Fig. 8A). In contrast, rhFSH failed to induce 10-week-SCs to gain an ability to incorporate BrdU after 48-hour treatment (Fig. 8B).

Figure 8.

FSH induces cell proliferation only on 20-day-SCs (A) but not on 10-week-SCs (B). 20-day-SCs and 10-week-SCs were cultured for 18 days and 9 days, respectively, prior to a 2-day treatment with rhFSH and subsequently detection of DNA replication by BrdU incorporation. Negative controls (−rhFSH) were parallel cultures that were not treated with the peptide hormone. Results shown are representative of 2 replicate experiments.

Proteomic analyses

SWATH mass spectrometry was performed on equal amounts of proteins in spent culture medium of 20-day-SCs and 10-week-SCs in order to determine whether the distribution of each secreted protein, especially of those involved in cell proliferation, was different between the 2 cultures. There were 845 proteins altogether that were commonly present in spent medium of the 2 cultures (see the complete list in Reference (45)), but only 144 proteins showed significant differences in distribution. Eleven groups of 70 proteins with specific properties, having more than twofold changes between the 2 cultures were selected and displayed in Table 2 and also in full in Reference (45). All or the majority of proteins in Groups I to VI were in higher amounts in spent medium of 20-day-SCs, compared with 10-week-SCs. However, the opposite was observed with proteins from Groups VII to XI.

Table 2.

Select Proteins in Culture Spent Medium With Log₂ Fold Change More Than 1 Compared Between Those From Sertoli Cells Isolated From 20-Day-Old Versus Those From 10-Week-Old Mice

Group	Protein name (UniProt ID)	Gene name	No. peptides	P value	Log2 fold change*
I. Hormones	Müellerian-inhibiting factor (aka anti-Müellerian hormone (P27106))	Amh	6	0.00618	–4.33820
	Inhibin alpha chain (Q04997)	Inha	7	0.01728	-2.75445
	Inhibin beta A chain (Q04998)	Inhba	7	0.04676	-3.35450
	Follistatin-related protein 4 (Q5STE3)	Fstl4	4	0.01377	-3.83380
II. Proteins involved in cell division	Cell division control protein 42 (P60766)	Cdc42	1	0.01947	-2.70454
	Rho GDP-dissociation inhibitor (Q99PT1)	Arhgdia	3	0.02853	1.63339
III. Collagen isoforms	Collagen alpha-2(VI) chain (Q02788)	Col6a2	11	0.00131	-2.85537
	Collagen alpha-1(I) chain (P11087)	Col1A1	20	0.00477	-3.29491
	Isoform 3 of collagen alpha-1(XVIII) chain (P39061)	Col18a1	6	0.00727	-4.60532
	Collagen alpha-2(V) chain (Q3U962)	Col5A2	19	0.00782	-2.69540
	Collagen alpha-2(I) chain (Q01149)	Col1A2	20	0.00779	-4.77428
	Collagen alpha-1(V) chain (O88207)	Col5a1	20	0.01037	-3.34629
	Collagen alpha-1(XVI) chain (Q8BLX7)	Col16a1	5	0.02900	-2.73981
	Collagen alpha-1(VIII) chain (Q00780)	Col8a1	1	0.04020	-2.55596
	Collagen alpha-1(XV) chain (O35206)	Col15a1	19	0.04056	-3.65484
	Collagen alpha-1(III) chain (P08121)	Col3a1	20	0.04216	-1.40279
IV. Proteoglycans and associated proteins	Chondroitin sulfate proteoglycan 4 (Q8VHY0)	Cspg4	11	0.01417	-2.99983
	Versican core protein (Q62059)	Vcan	7	0.01419	-2.07323
	Inter-alpha-trypsin inhibitor heavy chain H2 (Q61703)	Itih2	2	0.01888	1.31928
	Brevican core protein (Q61361)	Bcan	1	0.020401	-3.32063
	Isoform 2 of Tenascin (Q80YX1)	Tnc	18	0.022665	-3.4328
	Syndecan-4 (O35988)	Sdc4	11	0.03052	-2.41226
V. Cytokine binding protein	Latent-transforming growth factor beta-binding protein (Q8CG19)	Ltbp1	7	0.00465	-1.98310
VI. Extracellular proteins involved in cell adhesion, differentiation, outgrowth and plasticity	Matrix Gla protein (P19788)	Mgp	6	0.00073	-5.34998
	Osteopontin (P10923)	Spp1	20	0.00087	-2.56924
	Neuronal pentraxin-2 (O70340)	Nptx2	2	0.00134	-3.57135
	Semaphorin-3D (Q8BH34)	Sema3d	6	0.00463	-3.25079
	Spondin-1 (Q8VCC9)	Spon1	7	0.00615	2.55606
	Endosialin (aka CD248) (Q91V98)	Cd248	2	0.02014	-3.68950
	Isoform C of Fibulin-1 (Q08879)	Fbln1	3	0.03972	-2.54080
VII. Proteins involved in oxidation and reduction	Glutathione peroxidase 3 (P46412)	Gpx3	5	0.00610	2.72026
	Peroxidasin homolog (Q3UQ28)	Pxdn	15	0.01958	-2.80296
	Glutathione S-transferase Mu3 (P19639)	Gstm3	1	0.01266	4.53691
	Lysyl oxidase homolog (Q9Z175)	Loxl3	4	0.01266	-2.40445
	Peroxiredoxin-2 (Q61171)	Prdx2	3	0.01644	2.65851
	Glutathione S-transferase A4 (P24472)	Gsta4	4	0.02179	3.28447
	Glutathione S-transferase Mu 1 (P10649)	Gstm1	20	0.02228	3.89420
	Thioredoxin (P10639)	Txn	2	0.02352	3.40633
	Peroxiredoxin-6 (O08709)	Prdx6	11	0.02405	1.58273
	Glutathione reductase, mitochondrial (P47791)	Gsr	2	0.02853	1.57829
	Glutathione S-transferase omega (O09131)	Gsto1	1	0.03070	1.38964
	Glutathione S-transferase P 1 (P19157)	Gstp1	6	0.04076	1.67904
	Glutathione S-transferase A1 (P13745)	Gsta1	2	0.04660	2.87813
	Glutathione S-transferase Mu 2 (P15626)	Gstm2	9	0.04659	2.64716
	Thioredoxin-dependent peroxide reductase (P20108)	Prdx3	4	0.04783	1.66743
	Peroxiredoxin-5, mitochondrial (P99029)	Prdx5	5	0.04927	1.59490
VIII. Enzymes involved in glycolysis and Krebs cycle	Isocitrate dehydrogenase [NADP] cytoplasmic (O88844)	Idh1	12	0.00117	3.42142
	NADP-dependent malic enzyme (P06801)	Me1	18	0.00139	2.29365
	Pyruvate kinase PKM (P52480)	Pkm	18	0.00162	3.33252
	L-lactate dehydrogenase A chain (P06151)	Ldha	9	0.00245	2.55040
	Alpha-enolase (P17182)	Eno1	18	0.00282	1.92675
	Fructose-bisphosphate aldolase A (P05064)	Aldoa	13	0.00347	2.06011
	Malate dehydrogenase, cytoplasmic (P14152)	Mdh1	5	0.00371	1.94682
	Triosephosphate isomerase (P17751)	Tpi1	6	0.00601	2.55414
	Pyruvate kinase PKLR (P53657)	Pklr	1	0.00850	3.37595
	Phosphoglycerate kinase 1 (P09411)	Pgk1	10	0.02363	1.78430
	Glyceraldehyde-3-phosphate dehydrogenase (P16858)	Gadph	9	0.02846	2.11963
	Malate dehydrogenase, mitochondrial (P08249)	Mdh2	7	0.03713	1.53668
IX. Proteins involved in lipid metabolism	Fatty acid-binding protein, heart (P11404)	Fabp3	7	0.00403	4.15167
	Phospholipase D3 (O35405)	Pld3	2	0.01251	2.60316
	Phosphatidylethanolamine-binding protein 1 (P70296)	Pebp1	7	0.01971	2.47752
	Adrenodoxin, mitochondrial (P46656)	Fdx1	4	0.03037	2.23969
	Hydroxymethylglutaryl-CoA synthase, mitochondrial (P54869)	Hmgcs2	3	0.04082	1.83120
	Coatomer subunit gamma-1 (Q9QZE5)	Copg1	1	0.04182	2.45946
X. Proteins involved in hormone metabolism	Prostaglandin-H2 D-isomerase (O09114)	Ptgds	4	0.00599	4.24163
	Estradiol 17 beta-dehydrogenase 5 (P70694)	Akr1c6	1	0.01427	3.30893
	Adrenodoxin, mitochondrial (P46656)	Fdx1	4	0.03037	2.23969
XI. Cytoskeletal proteins	Tubulin beta-5 chain (P99024)	Tubb5	18	0.00662	2.07185
	Tubulin alpha-1A chain (P68369)	Tuba1a	16	0.00785	2.13984
	Vimentin (P20152)	Vim	19	0.02959	1.52205

*log₂ fold change = log₂(a) - log₂(b) = log₂(a/b); a and b are ion counts of Sertoli cells isolated from 10-week-old and 20-day-old animals, respectively. The value will be +, when a value is higher than b and will be −, when b value is higher than a, and will be > 1 and < −1, respectively, when the change was higher than twofold. Raw fold changes were also calculated and shown in Reference (45).

Note that most or all proteins in Groups I-VI have higher ion counts in samples from 20-day-old mice, whereas the opposite data are found in Groups VII-XI.

Of particular interest were proteins involved in cell proliferation/division including AMH (89) and CDC42 (90). The presence of inhibin βA subunit in the spent medium also suggested that the activin A dimer could be expressed by Sertoli cells (91). Activin has been previously shown to stimulate Sertoli cell proliferation (35, 92). In addition, collagens and proteoglycans have been reported for their positive influence on cell proliferation, cell migration, and process extension (93–95). Therefore, the higher levels of these proteins in spent medium of 20-day-SCs (Table 2) corroborated the cell proliferation activity of these Sertoli cells (Figs. 6 and 7).

Proteins in Groups VIII to XI with higher distribution in medium secreted from Sertoli cells derived from adult mice (Table 2) are proteins/enzymes involved in cellular metabolism (in glycolysis and Krebs cycle and lipid and hormone metabolism), survival and functionality (oxidation and reduction) and structural stability (cytoskeletal proteins). These proteins are expected to belong to differentiated cells, such as 10-week-SCs.

To confirm the proteomic results, immunoblotting was performed on select proteins present in the culture medium of 20-week-SCs and 10-week-SCs. Immunoblotting signals of 2 proteins in the 2 cultures with no significant differences in distribution as revealed by SWATH MS, namely ABP (SHBG) and prosaposin (PSAP) (45), are shown in the top row of Fig. 9A. The intensity of the ABP band was only slightly lower in culture medium from 10-week-SCs. For PSAP, the intensity of the unprocessed 70 kDa band was the same in cultures of Sertoli cells isolated from mice of the 2 ages, although the intensity of the processed bands of 53 kDa and 34 kDa was somewhat lower in medium from 10-week-SCs. In contrast, drastic differences in the intensity of AMH and GPX3 bands were observed (bottom row, Fig. 9A). While the 55 kDa band of AMH (the active N-terminal pro-region form (96) was similar in culture medium from 20-day-SCs and 10-week-SCs, the intensity of the 49 kDa AMH band (presumably the processed form of the 55 kDa band) was much higher in medium from 20-day-SCs. In contrast, GPX3 band was detected only in medium from 10-week-SCs (Fig. 9A).

Figure 9.

Immunoblotting of proteins from cultures of 20-week-SCs and 10-week-SCs. (A) proteins secreted into medium (PSAP, ABP, AMH, GPX3). (B) cellular proteins (AMH, CDC42, p27^Kip1, β-tubulin (TBB). Proteins present in spent medium of Sertoli cell cultures (30 μg for ABP, 20 μg for AMH and 10 μg each for PSAP and GPX3) were used to load into each lane for PAGE/immunoblotting. For Sertoli cellular proteins, 20 μg each were used for immunoblotting detection of AMH, CDC42 and TBB and 40 μg for p27^Kip1. Arrowheads point the protein bands with their molecular mass indicated in kDa.

To further confirm that AMH was expressed at a higher level by 20-day-SCs, equal amounts of proteins extracted from 20-day-SCs and 10-week-SCs were also subjected to immunoblotting. While the levels of the inactive full-length form of AMH (70 kDa) (96) appeared to be similar in 20-day-SCs and 10-week-SCs, the amount of the active 55 kDa form of AMH was apparently higher in 20-day-SCs (Fig. 9B). This difference in AMH amount would be more pronounced if it was expressed per cell, since the total proteins per 20-day-SCs were only one-fourth of those from 10-week-SCs (302 ± 34 pg/cell versus 1344 ± 215 pg/cell for 20-day-SCs and 10-week-SCs, respectively; n = 3 sets of experiments; P < 0.001). The levels of CDC42 in 20-day-SCs were also higher than those in 10-week-SCs (Fig. 9B). In contrast, the levels of p27^Kip1 (aka cyclin-dependent kinase inhibitor 1B [CDKN1B]), which is known to be inversely correlated with Sertoli cell proliferation (97), were higher in 10-week-SCs (Fig. 9B). However, the amounts of the structural protein β-tubulin (TBB) were present in a similar distribution in Sertoli cells of 20-day-SCs and 10-week-SCs.

Discussion

Various in vivo studies indicate that rat and mouse Sertoli cells cease to proliferate, upon reaching maturation at around Day 16 after birth, as discussed in the Introduction. Therefore, properties of primary cultures of Sertoli cells isolated from 20-26-day-old rats/mice are considered to be similar to those of cultures of Sertoli cells derived from adult animals, and cultured Sertoli cells from 20-day-old rats/mice have long been used for research studies as surrogates of adult Sertoli cells. Our report herein demonstrated that mouse 20-day-SCs and 10-week-SCs had different cell biology and morphological properties as well as different lipidomic and proteomic profiles, and the results from cell biology and morphological studies corroborated those from lipidomic and proteomic analyses. Most importantly, while mouse 10-week-SCs no longer divided as expected (13, 29), 20-day-SCs still proliferated in culture.

Isolation of a pure population of Sertoli cells from adult mouse/rat testes is challenging, since Sertoli cells constitute only 3% to 5% of total seminiferous tubule cells (28–31), and only a limited number of publications have described their isolation and culture procedures (38–41, 43, 44). Nonetheless, isolation procedures of rat/mouse Sertoli cells from adult males share common steps with those from animals 20 days of age or younger (48, 49, 98, 99). Seminiferous tubules are digested with enzymes including collagenase, trypsin, hyaluronidase, and DNase to generate a mixture of loose Sertoli cells and testicular germ cells. The majority of myoid cells were also removed from seminiferous tubules in between the enzyme digestion steps by treatment with 1 M glycine. When loose Sertoli cells and testicular germ cells are plated onto a culture dish, adherent Sertoli cells attach to the substratum, thus initiating their culture. In contrast, germ cells are not adherent, and in most protocols, their removal is expedited by their rupture following treatment of the culture with a hypotonic solution (100). In previous reports, partial separation of Sertoli cells from testicular germ cells through differential sedimentation in 2% to 3% BSA solution was also included for Sertoli cell isolation from adult rats (41, 44). However, our attempt to use this step not only failed to improve the purity of Sertoli cells but also decreased the yield. In addition, we observed that exposure of Sertoli cells in culture (both 20-day-SCs and 10-week-SCs) to a hypotonic solution did cause transient appearance of intracellular vacuoles (45). Although these vacuoles disappeared in due time, it was unclear whether these treated Sertoli cells changed their metabolism. In fact, the suspension of Sertoli cells, isolated from adult rats, cannot attach to a substratum for culturing after treatment with a hypotonic solution (38, 43). In our experience, testicular germ cells could be simply washed away from the adherent Sertoli cell culture during medium replacements.

Purification of Sertoli cells from testicular germ cells by the specific property of Sertoli cells to adhere to DSA lectin coated on a culture dish has also been described (101). While this procedure is effective in increasing the yield of Sertoli cells, it remains to be seen whether adult Sertoli cells isolated from this method have the same biochemical and physiological properties as adult Sertoli cells isolated by our method that is not dependent on an extra reagent (i.e., lectin), physiologically unrelated to Sertoli cells.

The high purity and optimum yield of adult Sertoli cells may be attributed to a number of steps in our isolation protocol. First, the amounts of enzymes and their treatment times for a number of testes were optimized empirically to yield loose cells from > 90% seminiferous tubules. We also found that the use of collagenase as the first enzyme to disperse seminiferous tubules from each other allowed a better digestion of tubules by trypsin (the second major enzyme) into small pieces. This in turn likely enhanced the production of loose cells from tubule pieces during the second digestion by collagenase (see Materials and Methods). The removal of particulates (likely small pieces of seminiferous tubules that were not well-digested by enzymes) by filtering through a 100 μm cell strainer also yielded a homogenous loose cell suspension (containing Sertoli cells), which would allow better culture plating. In addition, the exclusion of the majority of round spermatids and heads plus tails of testicular sperm and elongated spermatids into the supernatant by 50g centrifugation resulted in a higher distribution of Sertoli cells in the pellet, the suspension of which was used for culture plating.

Another important factor to obtain a high yield and purity of Sertoli cells from adult mice was to plate the optimal number of the mixture of loose Sertoli cells and testicular germ cells into the Sarstedt 10-cm culture dish, which was selected based on its highest affinity for Sertoli cells. This number of these mixed cells was empirically determined to be 30 million in 10 mL medium. At this mixed cell concentration, there were enough Sertoli cells to adhere and occupy the substratum and contact each other to form tight junctions. On the other hand, the concentration of germ cells was not high enough to impede Sertoli cells to settle onto the plate surface, and without the ability to adhere to the plate, the germ cells could be removed during medium changes. The purity of our 10-week-SCs was therefore consistently higher than 90%; the yield was sufficient for not only cell biology but also biochemical studies (Fig. 1B and 1C).

Comparing 20-day-SCs and 10-week-SCs in parallel revealed that the 2 cultures did not have identical properties. While they possessed common Sertoli cell features, namely possession of tripartite nuclei (45), tight junction proteins (claudin-11 and ZO-1) (Fig. 2) and lipid droplets (Fig. 4), the levels of claudin-11 and ZO-1 and lipid droplets were lower in 20-day-SCs (Figs 2 and 4). The length and width of Sertoli cells of 10-week-SCs were also 1.5X of those of 20-day-SCs (45). The most pronounced difference was that 20-day-SCs were still dividing, in particular, from Day 3 to Day 9 in culture, whereas 10-week-SCs no longer proliferated throughout the 9-day culture period. We demonstrated cell proliferation in 20-day-SCs directly by cell counting (Fig. 6A) and confirmed the results by the presence of the metaphase plate on culture Day 4 (Fig. 6B) and nuclear incorporation of BrdU from culture Day 3 to Day 10 (Fig. 7). However, the increase in cell number and BrdU incorporation was no longer observed in 20-day-SCs when the culture was prolonged to Day 15 (Figs 6 and 7). Nonetheless, the cell proliferation signaling pathway was likely still functioning, since FSH treatment of 20-day-SCs on culture Day 18 induced BrdU re-incorporation (Fig. 8). In contrast, BrdU incorporation was not observed in 10-week-SCs throughout culture Day 4 to Day 11 and even after FSH treatment on culture Day 9. Our result is dissimilar to a previous report (39), which describes that ~15% and 25% of adult Sertoli cells incorporate BrdU on culture Day 7 and Day 13, respectively (39). This disparity may be attributed to differences in the isolation/culture conditions. First, our 10-week-SCs were never exposed to a hypotonic solution, whereas this treatment was applied to their Sertoli cell cultures (39), and it was possible that the BrdU incorporation observed may reflect DNA repair. Second, our 10-week-SCs had already made contact with one another by Days 6 and 7 in culture (Fig. 3) in contrast to cultures of adult Sertoli cells in the study by Ahmed et al (39), which still showed empty spaces between each other. On the other hand, our observation that FSH failed to induce proliferation of 10-week-SCs agrees with results described by Griswold et al (102), showing no ³H-thymidine incorporation into cultures of Sertoli cells isolated from 8.6-week-old and older rats.

Assembly of tight junctions is correlated with an arrest in cell proliferation (14, 103). ZO-1 is known to capture ZONAB, a cell proliferation promoting transcriptional factor. Being sequestered to the tight junctions, ZONAB would no longer be able to stimulate cell proliferation (104). Consequently, the correlation between the high immunofluorescence signals of claudin-11 and ZO-1 and the lack of proliferation in 10-week-SCs is not surprising (Figs 2 and 6). In contrast, the much lower signals of claudin-11 and ZO-1 in 20-day-SCs indicated that the tight junctions were not fully developed and ZONAB would still be free to stimulate cell division in 20-day-SCs. Our proteomics and immunoblotting results further revealed higher levels of cell-proliferation-associated proteins, including CDC42 and AMH (89, 90), in spent medium and/or cell extracts of 20-day-SCs, compared with 10-week-SCs. CDC42 has an important role in spindle formation and orientation during mitosis (90), but its activity can be decreased by Rho GDP-dissociation inhibitor (105). The higher levels of CDC42 but lower amounts of Rho GDP-dissociation inhibitor in spent medium of 20-day-SCs mice (Table 2) corroborate our observation that 20-day-SCs were still proliferating.

The presence of inhibin α-subunit and inhibin βA subunit in the culture medium indicated that inhibin A dimer and activin A dimer could be expressed by Sertoli cells (91), with higher amounts from 20-day-SCs (Table 2) than 10-week-SCs. Activin has been shown for its stimulation on Sertoli cell proliferation (35,92) and could be one factor in enhancing proliferation of 20-day-SCs. In contrast, inhibin and follistatin-related protein 4 are expected to antagonize the effects of FSH and activin on Sertoli cell proliferation (91, 106–108). The higher levels of inhibin and follistatin-related protein 4 in 20-day-SC medium may reflect their important roles in vivo in counteracting FSH effects on Sertoli cell proliferation around this age of the animal (13, 14). Regardless, both inhibin and follistatin-like protein 4 may also lower the activity of activin-induced proliferation in 20-day-SC cultures to a certain extent, although not to a zero level.

Our observation that 20-day-SCs continued to proliferate indicates that these cultured Sertoli cells are under different regulations from those of Sertoli cells inside the testis of 20-day-old mice, where proliferation has already terminated (13). Besides inhibin and follistatin, thyroid hormone has been documented to inhibit cell proliferation in vivo through its induction of p27^Kip1 expression. p27^Kip1 (CDKN1B) is a cell cycle inhibitor protein, thus slowing down cell division (97, 107, 109). In vivo, the expression of p27^Kip1 is well observed in Sertoli cells of 16-day-old and older mice due to the systemic exposure to thyroid hormone (109, 110). In culture, Sertoli cells are no longer exposed to thyroid hormones and our results revealed that p27^Kip1 was present at a minimal level in 20-day-SCs, in contrast to its appreciable amount in 10-week-SCs (Fig. 9B). These results would explain in part the cell proliferation activity and the lack thereof in 20-day-SCs and 10-week-SCs, respectively. In the same vein, the higher level of LTBP1 in 20-day-SCs may be beneficial for their proliferation. LTBP1 is an extracellular protein complex, covalently linked to LAP (latency associated protein), which in turn noncovalently captures TGF-β from exerting its inhibitory effect on cell growth (111–113).

Previous results indicate that Sertoli cells isolated from 23- to 27-day-old rats and cultured for 1–2 days as aggregates also proliferate after being transplanted underneath the mouse or rat kidney capsule (114). Together with our result, this implicates that mouse and rat Sertoli cells are not terminally differentiated, in agreement with the previous observation in cultured adult Sertoli cells (14). Presumably, the regulatory factors on proliferation exist only in vivo. They can be systemic (such as thyroid hormone) or architectural to the seminiferous tubules (such as spermatogonia, developing germ cells, myoid cells and the basement membrane). Once outside the body, Sertoli cells by themselves in culture could therefore proliferate. Regardless, the degree of proliferation in vitro is more obvious with Sertoli cells isolated from 20- to 27-day-old-mice and rats (114). Our reports herein also indicate other different parameters between 20-day-SCs and 10-week-SCs and therefore, the use of cultures of Sertoli cells derived from mice 20 days of age or younger in place of cultures of Sertoli cells isolated from adult animals should be avoided. Our method described herein to isolate and culture Sertoli cells from adult mice with > 90% purity should make it possible to directly use cultures of adult Sertoli cells in research studies.

Glossary

Abbreviations

α-SMA

alpha-smooth muscle actin

20-day-SCs

cultures of Sertoli cells isolated from 20-day-old mice

10-week-SCs

cultures of Sertoli cells isolated from 10-week-old mice

ABP

androgen binding protein

AMH

anti-Mullerian hormone

BrdU

bromo-2-deoxyuridine

BSA

bovine serum albumin

CDC42

cell division cycle protein 42

CE

cholesteryl ester

Cer

ceramide

COUP-TFII

COUP transcription factor 2

ECL

enhanced chemiluminescence

FSH

follicle-stimulating hormone

GPX3

glutathione peroxidase 3

HRP

horseradish peroxidase

MRM

multiple reaction monitoring

MS

mass spectrometry

PAGE

, polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PC

phosphatidylcholine

PFA

paraformaldehyde

PE

phosphatidylethanolamine

PI

phosphatidylinositol

PSAP

prosaposin

rhFSH

recombinant human FSH

RT

room temperature

SBTI

soybean trypsin inhibitor

SC

Sertoli cell

SGG

sulfogalactosyl glycerolipid

SHBG

sex hormone binding globulin

SM

sphingomyelin

SWATH

sequential window activation of all theoretical masses

TAG

triacylglycerol

TBB

β-tubulin

WT1

Wilms tumor protein 1

Financial Support: This work was supported by Canadian Institutes of Health Research (CIHR, MOP-119438 and MOP-84420, given to NT). KK was a recipient of a scholarship from Development and Promotion of Science and Technology Talented Project (DPST), Ministry of Education, Thailand, as well as CIHR-Strategic Training Initiative in Health Research (STIRH), Canada, with the training in Reproduction, Early Development and the Impact on Health (REDIH).

Additional Information

Disclosure Summary: The authors have nothing to disclose.