Quantification of stem cell factor mRNA levels in the rat testis: usefulness of clusterin mRNA as a marker of the amount of mRNA of Sertoli cell origin in post pubertal rats.
Spermatogenesis is a complex cellular process regulated by gonadotrophins and local cell-cell interactions. Stem cell factor (SCF) is one of the paracrine factors, produced by the Sertoli cells, involved in the local regulation of spermatogenesis. Measurement of its testicular level is important for addressing its role in testis physiopathology. However, the relative cell composition of experimental and pathological testis samples may lead to misinterpretation in relating SCF mRNA levels to the amount of RNA extracted from the whole tissue sample. Taking into account the relative RNA content of Sertoli cell origin should provide more significant data. In the present study, three sets of experiments were intended for modifying the proportion of RNA of Sertoli cell origin in RNA extracted from whole testis tissue samples: during postnatal development; following methoxy-acetic acid (MAA) administration; and after injecting a long-acting gonadotrophin-releasing hormone agonist (GnRHa). In a first step, we demonstrated clusterin mRNA level stability in purified Sertoli cell preparations between 20 days and adulthood, and following MAA or GnRHa treatment. In a second step, we used a competitive RT-PCR assay to measure SCF and clusterin mRNA levels and expressed the amount of SCF mRNA relative to the amount of clusterin mRNA under the above experimental conditions. The SCF/clusterin mRNA level ratio was found to remain roughly stable from 20 days post-partum to adulthood; i.e. during the development of spermatogenesis. MAA administration led to an overall increase in the SCF/clusterin mRNA level ratio between 7 and 14 days after administration, consistent with the replenishment of the testis with pachytene spermatocytes and round spermatids. Conversely, after long-acting GnRHa injection, the SCF/clusterin mRNA level ratio decreased only slightly from day 21 onward. Hence, the present studies indicate that, under physiopathological conditions, the amount of clusterin mRNA is a good marker of the amount of RNA of Sertoli cell origin in testis samples at day 20 or later; different experimental alterations of spermatogenesis are associated with different patterns of SCF mRNA levels; the relationship between FSH and SCF in vivo is not as simple as that described in vitro.
UMR INSERM 418/INRA 1245, Université Claude Bernard Lyon 1, Hôpital Debrousse, 69322 Lyon, France.
Ingrid
I
Plotton
Pascale
P
Sanchez
Marie Hélène
MH
Perrard
Phillipe
P
Durand
Hervé
H
Lejeune
D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D051152:Clusterin; D005819:Genetic Markers; D006023:Glycoproteins / Q000235:genetics*; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D018832:Molecular Chaperones / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012708:Sertoli Cells / Q000737:chemistry; D012741:Sexual Maturation / Q000502:physiology*; D019089:Stem Cell Factor / Q000032:analysis / Q000235:genetics*; D013737:Testis / Q000737:chemistry*
D051152:Clusterin; D005819:Genetic Markers; D006023:Glycoproteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D019089:Stem Cell Factor