Generation of Theg deficient mice to characterise its role in spermatogenesis

-/-length Theg was used as a probe in Northern blot analysis, we observed a signal in testicular protamines (Prm1, Prm2 and Prm3) (Balhorn et al., 1984; Schlüterand Engel, 1995; Schlüter et al., 1996) and ADAM family proteins (Wolfsberg et al., 1995a; Wolfsberg et al., 1995b).

Different functions have been suggested for the nuclear proteins expressed in haploid germ cells, including nuclear shaping, histone removal, transcriptional repression, chromatin condensation, and, most recently, repair of the DNA strand breaks that normally transiently occur during the removal of the nucleosomes. (Zhao et al., 2001; Caron et al., 2001;

Kierszenbaum, 2001)

4.1.2 Generation of Theg deficient mice to characterise its role in spermatogenesis

The temporal-spatial nuclear expression of Theg in round spermatid cells suggests that Theg might play an important role during a successful differentiation of the male germ cells. To elucidate the function of Theg in mice, a targeted mutation of the gene was generated deleting most of the C-terminal of the protein. We deleted a genomic locus of Theg that included exon 4 to exon 7 of the gene, encompassing important nuc

homologous recombination in ES cells (figure 3.10 and 3.13). The heterozygous animals appeared phenotypically normal and were fertile, therefore haploinsufficiency of Theg allele have no effect in mice. The homozygous mice for Theg deleted allele were generated in two different genetic backgrounds namely, in inbred strain 129X1/SvJ and in hybrid strain C57BL/6J x 129X1/SvJ.

In our Theg deleted knock-out mice, we don’t expect a Theg transcript to exist, as the truncated transcript which will result in the knock-out mice will be

physiological condition due to lack of 3’ untranslated region of the transcript. When Northern blot was performed using a Theg cDNA consisting of exons 3, 4, 5, 6, and 7, no signal was detected in testicular RNA from Theg , in heterozygous mice the intensity of signal was weaker then that of testicular RNA of wild type mice (figure 3.15A). However when full

RNA from Theg^-/- which was weaker compared to that of wild type testicular RNA (figure 3.15B). Further in Northern blot analysis with Neomycin as probe; we detected two bands in Theg^+/- and in Theg^-/- testicular RNA but no signal was detected in testicular RNA derived from wild type mice (figure 3.15D). This observation can be explained by the fact that during the generation of knock-out mice, a Neomycin cDNA under the control of Phosphoglycerate kinase promoter was introduced into mouse genome by homologous recombination, therefore one band corresponds to the endogenous transcript, the second transcript might be as a result of fusion of Theg truncated transcript with Neomycin cassette. In order to determine whether indeed a fusion transcript exists in Theg^-/- mice we performed RT-PCR with two different pairs of primers namely mTHEG1/NeoRI and mTHEG1/mTHEG2 on testicular RNA derived

usion transcript was generated in Theg^-/- mice could be because of herent property of Theg to undergo alternative splicing. Under normal circumstances Theg

n 3 is pliced out resulting in a Theg isoform, which only lacks exon 3. We believe that in Theg^-/-

ls (figure 3.20 J-L). These result clearly shows that generated Theg from Theg^-/- mice. A PCR product was amplified with mTHEG1/NeoRI, but no amplification product was observed with mTHEG1/mTHEG2 (figure 3.16). After sequence analysis of this amplified product we could confirm that indeed a fusion transcript exists in Theg^-/- mice. This fusion transcript is comprised of exon 1 and exon 2 of Theg fused with Neomycin transcript.

The reason that this f in

give rise to four different splice variant (as discussed in 4.1.1), in one instance exo s

mice perhaps near Neomycin cassette there is a cryptic splice acceptor site, which generated a splice fusion Theg isoform (figure 4.1) and since Neomycin cassette included polyadenylation sequence, the fusion transcript generated is stable in vivo.

The next obvious question is what is the relevance of this fusion transcript in Theg^-/- mice. As wild type Theg protein is predominately localised in the nucleus of round spermatids, therefore we sought out the intracellular localisation of fusion Theg protein.

Immunocytochemical staining of cellular suspension isolated from Theg^-/- testes showed a cytosolic localisation of Theg fusion protein (figure 3.20 C-F). This result was furthered supported by generated Gfp tagged Theg fusion protein, which is expressed only in cytoplasm of transfected NIH3T3 cel

fusion protein in Theg^-/- mice have lost its ability to localise predominately in the nucleus of round spermatid cells. However a rational question at this juncture, which still remains unanswered is what functional role (if any) this fusion protein play in Theg^-/- mice.

Phenotypically, homozygous mice from both genetic backgrounds (in inbred strain 129X1/SvJ and in hybrid strain 129X1/SvJ x C57BL/6J) appeared normal and were fertile.

5’ 3’

1 2 3 4 5 6 7 8

1 2 3 NE0 8

5’ 3’

1 2345 6 7 8 1 245 6 7 8 1 2 NE0

Thegmajor Thegminor Thegfusion

mTHEG1 mTHEG2

NeoR1

Figure 4.1: Schematic diagram showing alternative splicing in Theg gene, which in case of wild type allele give rise to Theg major and Theg minor transcript. However in case of mutant allele, alternative splicing in Theg gene give rise to Theg fusion transcript. mTHEG1, mTHEG2, and NeoR1 were the primers used for RT-PCR.

eviation from Mendelian mode of

al. (2001) d 129X1/SvJ background. However a point worth mentioning is, in both examples discussed When a phenotype-genotype correlation was made, we discovered that in genetic background C57BL/6J x 129X1/SvJ the Theg deleted allele showed d

inheritance (figure 3.17). The ratio of heterozygous and homozygous mice are reduced as compared to that of wild type mice, suggesting that during segregation of alleles, Theg deleted allele is disadvantageous to wild type allele. However the mode of inheritance in Theg deleted allele in genetic background 129X1/SvJ did not show any deviation from Mendelian inheritance. An inferior Theg deleted allele in genetic background C57BL/6J x 129X1/SvJ could be due to genetic heterogeneity of the mice. There are numerous reports, which showed that genetic heterogeneity causes difference in phenotype in knock-out mice. For example Nayernia et al. (2002) showed that Smcp^-/- (sperm mitochondria-associated cysteine-rich protein) mice in mixed genetic background (C57BL/6J x 129X1/SvJ) are fully fertile, while they are infertile in the 129X1/SvJ background, in an another report Adham et

showed that Tnp2^-/- (transition protein2) male mice were normal and fertile in mixed genetic background C57BL/6J x 129X1/SvJ, however they were totally infertile in the inbre

above, the mixed genetic background of C57BL/6J x 129X1/SvJ serves as an advantage to the deleted allele but in contrary in our case, genetic heterogeneity resulted in minor inferiority to Theg-deleted allele. It is very difficult to speculate a reason when interactions of nature of such complex genetic heterogeneity is involved.

In another observation we noticed that testes of Theg^-/- mice appeared smaller than the testes of wild type (littermates) mice. When the growth curve of the testes development was determined, we observed a minor but significant retardation in case of Theg^-/- mice (figure 3.18). However histological examination of the testis revealed that in Theg^-/- mice the occurrence of spermatogenesis was normal and also the number of spermatozoa present in the epididymis was comparable to that of wild type mice. The spermatozoa from Theg^-/- mice were also able to fertilise ooyctes in a normal fashion as determined by in vitro fertilisation (IVF) assay. The morphological appearance of Thegdeficient sperm under the microscope also appeared to be normal.

We were also unable to detect any significant defect in motility of Theg^-/- sperm, as determined by computer assisted semen analysis (CASA) system (CEROS version 10, Hamilton Throne Research) (figure 3.19). So from the results of Theg deleted knock-out mice, we can conclude that deleted Theg is a non-essential protein in mice and spermatogenesis lthough we observed some minor defects as discussed above however those abnormalities

During the course of time when Theg deletion knock-out mice were generated, Yanaka et al.

(2000) reported a transgenic mouse, where a foreign DNA (human PDE5A gene under the control of cytomegalovirus promoter) was integrated into Theg locus. In their mice, several copies of the trangene were integrated into Theg locus thus producing a deletion of about 20 kb in the genomic locus. This insertion deleted the complete open reading frame (ORF) of Theg gene and also deleted an additional 7 kb fragment upstream of Theg gene (figure 4.2).

Yanaka et al. (2000) named this deleted transgenic locus as Kisimo (ki). The male mice homozygous for ki locus were sterile and sterility arose from a defect in spermatogenesis.

They also reported that in ki/ki mice, there were virtually no spermatozoa in the lumina of seminiferous and epididymal tubules. On histological analysis of cross-sections of ki/ki testes, they discovered that elongated spermatids in the vicinity of the lumina of seminiferous tubules showed vacuolation and were occasionally phagocytosed by Sertoli cells (figure 4.3A&B).

progress to its completion in Theg knock-out mice and produces normal motile spermatozoa.

A

are subtle and does not effect Theg^- sperms ability to fertilise ooyctes.