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MTCH2 is differentially expressed in rat testis and mainly related to apoptosis of spermatocytes

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Abstract

MTCH2 has been described in liver as a protein involved in the intrinsic apoptotic pathway, although new evidence also associates this protein with cellular metabolism. In this work, the expression of MTCH2 in testis (an organ in which high levels of apoptosis normally take place as part of the spermatogenic process) is analyzed in rat, both at the mRNA and at the protein levels. Our results showed that MTCH2 was highly expressed in testis compared with other tissues and was differentially expressed according to developmental stage and testicular cell type. Protein expression was initially detected during the first spermatogenic wave at the time of meiosis onset and its levels increased in adulthood, with the highest expression levels being detected in meiotic prophase I. Specific differences in MTCH2 expression levels at the various stages of the adult seminiferous epithelium were also observed. Co-staining with TUNEL revealed a differential MTCH2 staining pattern in TUNEL-positive cells, mainly in dying primary spermatocytes, i.e., meiotic prophase I cells. Furthermore, upon mild hyperthermia (treatment shown to increase apoptosis in testis), MTCH2 levels rose concomitantly with a massive appearance of TUNEL-positive cells within the seminiferous tubules; these cells exhibited a differential MTCH2 distribution. Thus, MTCH2 is related to testicular apoptosis, especially during meiotic prophase.

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Abbreviations

CRBC:

Chicken red blood cells

DD:

mRNA differential display

MCs:

Mitochondrial carriers

MTCH2:

Mitochondrial carrier homolog 2

OMM:

Outer mitochondrial membrane

PCD:

Programmed cell death

PI:

Propidium iodide

tBID:

Truncated BID

TBST:

TRIS-buffered saline with Tween

TNF:

Tumor necrosis factor

UTR:

Untranslated region

RT-PCR:

Reverse transcription with the polymerase chain reaction

qRT-PCR:

Quantitative real-time reverse transcription polymerase chain reaction

HPRT:

Hypoxanthine guanine phosphoribosyl transferase

BSA:

Bovine serum albumin

DAPI:

4,6-Diamidino-2-phenylindole

TUNEL:

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

PBS:

Phosphate-buffered saline

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Acknowledgments

The authors thank Drs. Atan Gross and Yehudit Zaltsman (Weizmann Institute) for providing the anti-MTCH2 antibody, Dr. Ricardo Benavente (University of Würzburg) for the anti-SYCP1-Nt antibody and for critical reading of the manuscript and Dr. Gustavo Folle (IIBCE) for helpful comments.

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Author notes

  1. Andrés Goldman

    Present address: Department of Biological Regulation, The Weizmann Institute of Science, 234 Herzl Street, Rehovot, 7610001, Israel

Authors and Affiliations

  1. Departamento de Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avda. Italia 3318, Montevideo, 11600, Uruguay

    Andrés Goldman, Rosana Rodríguez-Casuriaga, Evangelina González-López, Carlos A. Capoano & Adriana Geisinger

  2. Servicio de Citometría de Flujo y Clasificación Celular (SECIF), Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay

    Federico F. Santiñaque

  3. Sección Bioquímica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

    Adriana Geisinger

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  1. Andrés Goldman
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  3. Evangelina González-López
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Correspondence to Adriana Geisinger.

Additional information

This work was supported by CSIC Uruguay (grant I + D C022 to A. Geisinger) and a postgraduate fellowship from ANII (Uruguay) to A. Goldman.

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Figure S1

a Expression pattern of Mtch2 RNA analyzed by RT-PCR with specific oligonucleotides over 18 or 40 PCR cycles. A parallel experiment with specific oligonucleotides for β-actin is shown as a control. For the 18-cycle experiment, all the reactions were performed in duplicate and run in adjacent lanes. b RT-PCR experiment (18 PCR cycles) comparing Mtch2 expression in highly enriched pachytene spermatocytes and round spermatids. Duplicate reactions were run in adjacent lanes. A parallel experiment for β-actin is shown as a control. Forward and reverse primers for Mtch2 were 5′-CCACCTAGAGGAGGATGAG-3′ and 5′-AAATGTCACTGTCCCTGCTC-3′, respectively, whereas primers for β-actin were 5′-CCATGTACGTAGCCATC-3′ and 5′-GTACCACCAGACAGCA-3′ (GIF 59 kb)

High resolution image (TIFF 2053 kb)

Figure S2

Co-staining for MitoTracker Red 580 (a, b) and MTCH2 (a’, b’) in adult rat testicular sections. Sections were counterstained with the DNA-specific fluorochrome DAPI (a’’, b’’). Merged images are shown right (a’’’, b’’’). b–b’’’ Higher magnification of a partial view of the images shown in a–a’’’. Bars 50 μm (a’’’), 20 μm (b’’’)(GIF 179 kb)

High resolution image (TIFF 7225 kb)

Figure S3

Immunoblot analysis of active caspase-3 in adult rat testes subjected to 0.5, 1, 3 and 6 h of mild hyperthermia (C control testes incubated at room temperature for the same time intervals). The same total protein amount was loaded into each lane. A Western blot for β-actin is shown as a control (GIF 9 kb)

High resolution image (TIFF 1056 kb)

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Goldman, A., Rodríguez-Casuriaga, R., González-López, E. et al. MTCH2 is differentially expressed in rat testis and mainly related to apoptosis of spermatocytes. Cell Tissue Res 361, 869–883 (2015). https://doi.org/10.1007/s00441-015-2163-2

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