Germ cells contribute to the function of the Sertoli cell barrier

Figures & data

Figure 1. Effects of germ cells on the Sertoli cell permeability barrier in vitro. Sertoli cells (SC) were seeded on Matrigel™-coated culture inserts at either 0.5 × 10⁶ cells/cm² or 1.2 × 10⁶ cells/cm² (time 0), and the first TER measurement was recorded 24 h thereafter. On day 3 in vitro, germ cells (GC) were isolated from the testes of 25- or 90- (adult) d-old rats, added to Sertoli cells (0.5 × 10⁶ cells/cm²) at a Sertoli:germ cell ratio of 1:3 (arrow) and cocultured for an additional 3 d. Germ cells isolated from 25-d-old rat testes consisted largely of spermatogonia and primary spermatocytes with few secondary spermatocytes and round spermatids. Successive passages through glass wool removed elongating/elongated spermatids and spermatozoa from 90-d-old germ cell isolations. TER measurements continued to be recorded at least once daily after the addition of germ cells. Controls consisted of culturing Sertoli cells at 0.5 × 10⁶ cells/cm² and 1.2 × 10⁶ cells in the absence of germ cells. Each error bar represents a mean ± SD of quadruple culture inserts. This experiment was performed four independent times using different batches of Sertoli and germ cells, with similar results obtained each time. For statistical analyses, each SC:GC coculture data point was compared with its corresponding SC culture (0.5 × 10⁶ cells/cm²) data point. *, P < 0.05; **, P < 0.01 (ANOVA followed by Tukey’s post hoc test).

Figure 2. Expression of BTB constituent genes by Sertoli and germ cells, and the testis. Germ cells (GC, n = 3) were isolated from 90-d-old rat testes and immediately harvested in either TRIzol^® reagent or lysis buffer. Sertoli cells (SC, n = 2) were isolated from 20-d-old rat testes, seeded onto Matrigel™-coated 12-well plates at 0.5 × 10⁶ cells/cm², hypotonically treated and cultured for 4 d before being harvested as described above. Testes (T, n = 2) were obtained from 90-d-old rats and used either for RNA extraction or lysate preparation. RT-PCR and immunoblotting were used to determine whether occludin (A), tricellulin (B), claudin-1 (C), claudin-5 (D), claudin-11 (E), JAM-A (F), CAR (G), FAK (H), AR (I), ZO-1 (J), and testin (K) were expressed by Sertoli and germ cells, and the testis. S-16 was used as a control in PCR experiments. Tables 1 and 2 list the primers and antibodies, respectively, that were used for this experiment. M, marker; bp, base pair; IB, immunoblotting.

Figure 3. Steady-state TJ and ES protein levels after coculture of germ cells with Sertoli cells. Sertoli cells (SC) were isolated from the testes of 20-d-old rats and seeded onto Matrigel™-coated 12-well plates at 0.5 × 10⁶ cells/cm². On day 3 in vitro, germ cells (GC) were isolated from the testes of 90-d-old rats, added to Sertoli cells at a Sertoli:germ cell ratio of 1:5 (time 0), and cocultured. Thereafter, cocultures (SC+GC) were terminated at different time points ranging from 0 h to 4 d. The control consisted of Sertoli cells cultured alone and terminated at identical time points. Lysates were obtained, and immunoblotting was performed as detailed in Materials and Methods and Table 2. Immunoblots assessed changes in the steady-state levels of occludin (A), tricellulin (B), claudin-5 (C), claudin-11 (D), JAM-A (E), CAR (F), FAK (G), AR (H), ZO-1 (I), N-cadherin (J), α-catenin (K), and β-catenin (L) after the addition of germ cells. To ensure equal loading of proteins, immunoblots were probed with an anti-actin antibody (A). Immunoblots were scanned to generate composite histograms (A–L). The relative protein level corresponding to Sertoli cells cultured alone at time 0 h (black bar) was arbitrarily set at 1. Each error bar represents a mean ± SD of three to five independent experiments using different batches of Sertoli and germ cells. For statistical analyses, each SC-GC coculture data point was compared with its corresponding control at 0 h (gray bar). * P < 0.05; **, P < 0.01 (Student’s t-test). Table 1 lists the antibodies that were used for this experiment.

Figure 4. Localization of occludin after coculture of germ cells with Sertoli cells. Sertoli cells (SC) were isolated from 20-d-old testes and seeded onto Matrigel™-coated glass coverslips at 0.05 × 10⁶ cells/cm². It is important to note that functional TJs and ESs were still capable of forming at this lower Sertoli cell density. On day 3 in vitro, germ cells (GC) were isolated from 90-d-old testes, added to Sertoli cells at a Sertoli:germ cell ratio of 1:5 (time 0), and cocultured for an additional 4 d at which time cells were immunostained for occludin (red) as detailed in Materials and Methods and Table 1. Sertoli (dotted-line ovals, C and D) and germ cell nuclei were visualized by DAPI (blue) staining. Corresponding images were merged (E and F). Brackets (E and F) denote the relative thickness of the immunoreactive fluorescent signal between adjacent Sertoli cells. The localization of occludin at the Sertoli–Sertoli cell interface is in agreement with previously published reports (see main text for references). Bar in A (also corresponds to B–F) = 20 μm.

Table 1. Primary antibodies used for immunoblotting and immunofluorescent staining

Antibody	Catalog No.	Lot No.	Host	Vendor	Working dilution
					IB	IF
Occludin	71–1500	913843A	Rabbit	Zymed/Invitrogen	1:250	1:50
Claudin-1	51–9000	20671575	Rabbit	Zymed/Invitrogen	1:250
Claudin-5	34–1600	558076A	Rabbit	Zymed/Invitrogen	1:250
Claudin-11	36–4500	728894A	Rabbit	Zymed/Invitrogen	1:250
Tricellulin	48–8400	500693B	Rabbit	Zymed/Invitrogen	1:250
JAM-A	36–1700	463126A	Rabbit	Zymed/Invitrogen	1:250
CAR	sc-15405	D1910	Rabbit	Santa Cruz Biotechnology	1:200
FAK	06–543	31701	Rabbit	Upstate	1:1000
AR	sc-816	H2009	Rabbit	Santa Cruz Biotechnology	1:200
ZO-1	61–7300	636050A	Rabbit	Zymed/Invitrogen	1:250
N-Cadherin	sc-7939	G2109	Rabbit	Santa Cruz Biotechnology	1:200
α-Catenin	sc-7894	G1007	Rabbit	Santa Cruz Biotechnology	1:200
β-Catenin	71–2700	60806848C2	Rabbit	Zymed/Invitrogen	1:250
Desmoglein-2	–	–	Rabbit	(Lie et al., 2010)	1:200
γ-Catenin	610254	74819	Mouse	BD Transduction Laboratories	1:1000
Actin	sc-1616	F0809	Goat	Santa Cruz Biotechnology	1:200

Abbreviations: IB, immunoblotting; IF, immunofluorescent staining.

Table 2. Primers used for RT-PCR

Gene	Primer sequence	Orientation	Position	Length (bp)	GenBank Accession No.	Tm (°C)	Cycle No.
Occludin	5′–CTGTCTATGC TCGTCATCG–3′ 5′–CATTCCCGAT CTAATGACGC–3′	sense antisense	202–220 476–495	294	NM_031329	58.0	30
Claudin-1	5′–CTTCTGGGTT TCATCCTGG–3′ 5′–CAGATTCAGC AAGGAGTCG–3′	sense antisense	22–40 201–219	198	NM_031699	54.5	30
Claudin-5	5′–GCCAACATCG TAGTCCG–3′ 5′–GTTCTTCTTG TCGTAATCGC C–3′	sense antisense	418–434 628–648	231	NM_031701	54.5	30
Claudin-11	5′–ATGGTAGCCA CTTGCCT–3′ 5′–TTAGACATGG GCACTCTTGG–3′	sense antisense	1–17 605–624	624	NM_053457	58.0	30
Tricellulin	5′–GTTACAGCCA CCATTCGG–3′ 5′–TTTGTAAGAG TTGGGTGGGT–3′	sense antisense	159–176 383–402	244	NM_001108936	54.6	30
JAM-A	5′–CTGAGAACGA CTCTGTCAAG–3′ 5′–AGTGAGGTGG ATGCTGACCT–3′	sense antisense	119–138 359–378	260	NM_031329	58.0	30
CAR	5′–CGCTCCTACT GTGCTTC–3′ 5′–CTTTCTGGTT ATCGGACGG–3′	sense antisense	5–21 181–199	195	NM_053570	54.0	30
FAK	5′–TCAAGTAGAC CAAGACATAG C–3′ 5′–CCTGAATGTT TGCTGGAT–3′	sense antisense	483–503 670–687	205	NM_013081	51.5	30
AR	5′–TGGAAGCACT GGAACATC–3′ 5′–CTCACCTTCC AACCCTTT–3′	sense antisense	740–757 940–957	218	NM_012502	54.0	30
ZO-1	5′–GCCTCTGCAG TTAAGCAT–3′ 5′–AAGAGCTGGC TGTTTTAA–3′	sense antisense	2788–2805 3019–3036	249	NM_001106266	58.0	30
Testin	5′–TGTGAAAATG ATGACAGGCT TT–3′ 5′–ACCATGGGTA ACATTAGATC CC–3′	sense antisense	264–285 510–531	268	NM_173132	58.0	30