Expression of Porcine FSH.BETA. Subunit Promoter-driven Herpes Simplex Virus Thymidine Kinase Gene in Transgenic Rats

Abstract

Abstract.A transgenic rat was established using the construct of porcine FSHβ subunit promoter, the -852/+10 bp region, fused to a Herpes simplex virus thymidine kinase (HSV-TK) gene. Integration of the transgene was confirmed by PCR of tail DNA. RT-PCR of total RNAs of the pituitary, gonad, cerebellum, liver, kidney, adrenal gland, prostate, and uterus revealed that FSHβ was only expressed in the pituitary. Analysis of the expression of reporter gene, HSV-TK, using two specific primer sets revealed that different transcripts were present in the pituitary and testis. The transcript initiated at the transcription initiation site of the porcine FSHβ gene was detected in the pituitary, and another within the TK gene was found in the testis, indi cating ectopic testis-specific e xpression. Immunohistochemistry of the pituitary glands of the transgenic rats for FSH and HSV-TK demonstrated that the FSH-producing cells also produced HSV-TK. The results indicated that the -852/+10 bp region of the FSHβ promoter contains an element(s) that determines the tissue-specific expression. We succeeded in producing FSHβ promoter-driven HSV-TK transgenic rats and were the first time to do so using an animal other than the mouse. The transgenic rats show male infertility that involves abnormal spermatogenesis. We also observed a decrease in the weight of the testis and epididymis, and both motile and living spermatozoa were absent in the epididymis. Consequently, the FSHβ-HSV-TK transgenic rat will provide a useful model for studies on FSH function and male infertility.

Accumulated HSV1-TK Proteins Interfere with Spermatogenesis through a Disruption of the Integrity of Sertoli-Germ Cell Junctions

“…Al-Shawi et al demonstrated that truncated TK polypeptides are translated mainly in the haploid spermatids by a cryptic TATA box-independent promoter and that the severity of male infertility depended on the level of enzymatic activity of HSV1-TK produced [5]. However, precise morphological analyses of the seminiferous tubules of HSV1-TK transgenic mice have not yet been reported.We previously reported that HSV1-TK transgenic rats also show male infertility by the ectopic spermatid-specific ectopic expression of HSV1-TK gene [7]. We have identified the testis-specific transcription start site of the HSV1-TK gene to produce two types of truncated TK polypeptides that retain enzymatic activity [8].…”

“…We previously reported that HSV1-TK transgenic rats also show male infertility by the ectopic spermatid-specific ectopic expression of HSV1-TK gene [7]. We have identified the testis-specific transcription start site of the HSV1-TK gene to produce two types of truncated TK polypeptides that retain enzymatic activity [8].…”

Molecular Cloning of LIM Homeodomain Transcription Factor <i>Lhx2</i> as a Transcription Factor of Porcine Follicle-Stimulating Hormone Beta Subunit (FSHβ) Gene

“…Hitherto, we demonstrated that the Fd2 region functions as a target site for multiple transcription factors [13] and that the porcine Fshβ promoter (-852/+10 b) is sufficient for gonadotrope-specific expression by generation of transgenic rats [16]. Later, as binding proteins for the Fd2, we cloned two pituitary transcription factors, i.e., the pituitary specific paired-like homeodomain transcription factor, PROP1 [14], and the paired-related homeodomain factor, PRX2 [15].…”

“…In addition, we have demonstrated that the −852/+10 b region is sufficient for the pituitary-specific expression of Fshβ [16]. PROP1 is essential for normal production of gonadotropins as well as for the development of PIT1 lineage cells in both humans and mice [17].…”

“…A similar region is also responsible for Pitx1, the homologue of both PITX2 [11] and FOXL2 [12]. Nevertheless, the mechanism underlying the cell/ tissue-specific control of Fshβ-expression remains unclear.We have previously reported that the Fd2 region (−852/−746 b of porcine Fshβ promoter) is a target for many nuclear proteins of the porcine anterior pituitary [13] as well as cloned Prop1 [14] and Prx2 [15], both of which belong to the homeodomain transcription factor family as an Fd2-binding protein.In addition, we have demonstrated that the −852/+10 b region is sufficient for the pituitary-specific expression of Fshβ [16]. PROP1 is essential for normal production of gonadotropins as well as for the development of PIT1 lineage cells in both humans and mice [17].…”