A 252 bp upstream region of the rat spermatocyte-specific hst70 gene is sufficient to promote expression of the hst70-CAT hybrid gene in testis and brain of transgenic mice.

The rat hst70 gene belongs to a heat shock hsp70 multigene family and its expression has been detected so far solely in spermatocytes. To investigate the cis-elements responsible for testis-specific expression of the hst70 gene we produced several lines of transgenic mice carrying fragments of the 5'-flanking regions of the hst70 gene fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Hybrid genes of series B were constructed such that, besides the 780 bp, 343 bp and 163 bp 5'-flanking region these plasmids contained no other sequences of the hst70 gene. In hybrid genes of series D the CAT gene was ligated to 343 bp and 252 bp 5'-flanking regions together with the 57 bp of the 5'-end nontranslated (leader) sequences of the hst70 gene. We found that in 780/B, 343/B, 343/D and 252/D adult mice the transgene was specifically and highly expressed in testes. In developing testes the high CAT activity appeared in transgenic mice aged 3 weeks and older. None of the three 163/B transgenic lines exhibited CAT activity in any tissue analyzed. In all CAT expressing lines a weak but significant CAT activity (up to 5% of that in testis) was detected also in the brain. RNase protection assay confirmed that the endogenous hst70 gene transcripts are present in testis as well as in brain of nontransgenic rats and mice. Our data show that the cis-regulatory sequences responsible for testis-specific and developmentally regulated expression of the hst70 gene are localized within the 252 bp region 5' to the gene and neither the 5'-end nor 3'-end nontranslated sequences of the gene are important for this specificity.

Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines.

To establish in vivo gonadal tumor models and permanent lines of gonadal somatic cells we produced transgenic (TG) mice expressing the Simian virus (SV) 40 T-antigens (T-ag), driven by 6 or 2.1 kilobase fragments of the mouse inhibin alpha-subunit promoter. Hitherto, altogether 44 TG mice, one of which carried the shorter transgene, have produced gonadal tumors. Two founder females expressing the longer transgene, KK1 and KK3, and three established TG mouse lines were studied in detail. Penetrance of the phenotype in IT6-M and IT6-F mouse lines was 100% (tumors/TG: IT6-M 22/22, IT6-F 14/14). The T-ag mRNA was strongly expressed in the gonads, adrenal glands, pituitary, and brain. The KK-1 and KK-3 ovarian tumor cells immunostained with anti-SV40 large-T antibody. The KK-1 cells possessed high-affinity LH receptors [equilibrium association constant (Ka = 7.8 x 10(10) liters/mol] and responded to human CG by elevated cAMP and progesterone production. Also FSH slightly stimulated their cAMP and estradiol production (P < 0.01). These cells expressed cytochrome P450arom and inhibin alpha mRNA, but not cytochrome P450c17 alpha. In conclusion, the KK-1 cells are immortalized luteinizing granulosa cells expressing endogenous gonadotropin receptors, steroidogenic enzymes, and inhibin alpha. These cells will be useful in studies on the molecular aspects of granulosa cell function. The present study indicates that the 6-kilobase fragment of the inhibin alpha promoter described in this article contains the elements directing tissue-specific expression in vivo and is useful for targeted expression of other genes in the gonads.

Gonadectomy permits adrenocortical tumorigenesis in mice transgenic for the mouse inhibin alpha-subunit promoter/simian virus 40 T-antigen fusion gene: evidence for negative autoregulation of the inhibin alpha-subunit gene.

We have developed a transgenic (TG) mouse model for gonadal tumorigenesis expressing the Simian virus 40 T-antigen (Tag) under the mouse inhibin alpha-subunit promoter. Gonadal tumors appear with 100% penetrance by the age of 5-8 months in the TG mice. When 1-month-old TG mice were gonadectomized, adrenal gland tumors were observed in each animal (12 females, 11 males) at the age of 6-8 months. No adrenal tumors were detected in gonadectomized non-TG mice (nine females, nine males) or in the intact TG mice (n > 100). The tumors appeared to originate from the X zone of the adrenal cortex. The TG mice with adrenocortical tumors had elevated serum levels of progesterone, estradiol, and immunoreactive inhibin (including dimeric forms), but corticosterone secretion was reduced. The lack of adrenal tumors in intact TG mice suggested that the tumorous gonads secrete factor(s) inhibiting adrenal tumorigenesis. As a candidate molecule, we studied the effects of inhibin, which was high in the serum of control females and TG females with ovarian tumors, as well as in TG males with testicular tumors. The DNA synthesis, as well as the levels of inhibin-alpha and Tag mRNA expression, were significantly reduced by recombinant human inhibin A in cell cultures derived from the adrenal tumors. In accordance, the expression level of inhibin-alpha mRNA in the normal adrenal gland was elevated 2 weeks after gonadectomy. These findings suggest that gonadal inhibin can down-regulate the expression of the inhibin alpha-subunit gene in the adrenal gland. When circulating inhibin is eliminated by gonadectomy, Tag expression and tumorigenesis are stimulated in the adrenal glands of the TG mice. The results demonstrate a novel mechanism of autoregulation in inhibin alpha-subunit gene expression.

The follicle-stimulating hormone (FSH) beta- and common alpha-subunits are expressed in mouse testis, as determined in wild-type mice and those transgenic for the FSH beta-subunit/herpes simplex virus thymidine kinase fusion gene.

Testicular expression of the endogenous FSH beta-subunit (FSH beta) and common alpha-subunit (C alpha) genes, as well as a Herpes simplex virus type 1 thymidine kinase (tk) transgene, driven by a 2.3-kilobase fragment of the bovine GSH beta promoter, were studied at messenger RNA and protein level in normal and transgenic mice. A major 3.8-kb species of FSH beta messenger RNA was demonstrated i the normal mouse testis by Northern hybridization. This was longer than the main 1.7-kb FSH beta transcript detected in the pituitary gland. Reverse transcription-polymerase chain reaction, followed by Southern hybridization, demonstrated FSH beta and tk expression in the pituitary gland and gonads of adult normal and transgenic mice, respectively. The C alpha expression was detected by reverse transcription-polymerase chain reaction in the pituitary gland and testis. During development, testicular transcription of the FSH beta and tk genes was initiated simultaneously a few days after birth. Immunocytochemistry of adult testes showed stage-specific positive reaction with FSH beta, C alpha, and tk antisera in the pachytene spermatocytes and type B spermatogonia, but not in Sertoli cells. Positive reaction with these antisera was also seen in the interstitial tissue. These results demonstrate testicular expression of the endogenous FSH subunit genes and confirm that the testicular expression of the FSH beta /tk transgene reflects or its subunits play a paracrine or autocrine role in the regulation of testicular function.

Novel expression of luteinizing hormone subunit genes in the rat testis.

The two gonadotropins, LH and FSH, are thought to be synthesized and secreted solely by the anterior pituitary. We present here evidence for expression of the LH beta and common alpha-subunit (C alpha) genes in the rat testis. The LH beta and C alpha-subunit messenger RNAs (mRNAs) were detected by reverse transcriptase-polymerase chain reaction in the rat testis and pituitary with primer pairs producing 247- and 199-base pair complementary DNA (cDNA) fragments, corresponding to nucleotides 154-400 of LH beta and nucleotides 250-448 of C alpha cDNA, respectively. The specificity of the cDNA species generated was verified by Southern hybridization using nested [32P]cDNA or oligonucleotide probes, and identity with the published rat LH beta and C alpha-subunit gene structures was determined by sequencing. The mRNA bands with specific hybridization to complementary RNA (cRNA) probes corresponding to nucleotides 154-368 of the rat LH beta cDNA and nucleotides 250-448 of the rat C alpha cDNA were found in the rat pituitary and testis by Northern hybridization. The major C alpha mRNA had a size of 0.8 kilobases (kb) in the pituitary and testis. The major LH beta transcripts were 0.8 and 2.7 kb in the pituitary and testis, respectively. To further characterize the larger testicular LH beta-subunit transcript, rapid amplification of the 3'-end of cDNA (3'-RACE) was performed using an oligo(deoxythymidine-17) adapter and a specific 5'-primer. Southern hybridization of the 3'-RACE product of rat testicular RNA with a LH beta [32P]cDNA probe had the same size as the 3'-RACE product of pituitary RNA. The pituitary and testicular RNAs were then cut into two segments using oligonucleotide-directed ribonuclease H digestion and subjected to Northern hybridization using a cRNA probe specific to the 5'-end segment. The digested 5'-end segments of the pituitary and testicular mRNAs were 0.4 and 2.3 kb, respectively, indicating that the testicular LH beta mRNA has a 1.9-kb 5'-extension, compared to the cognate pituitary mRNA. This was further verified by Northern hybridization using a cRNA probe corresponding to nucleotides -790 to -10 upstream of the pituitary initiation site of LH beta gene transcription. Specific hybridization of a 2.7-kb mRNA transcript was found in the rat testis, but none in the pituitary. Hence, the 3'-end polyadenalytion site of the LH beta mRNA is the same in rat pituitary and testis, and the different transcript sizes are due to a difference at the 5'-end.(ABSTRACT TRUNCATED AT 400 WORDS)

Suppression of gonadotropins inhibits gonadal tumorigenesis in mice transgenic for the mouse inhibin alpha-subunit promoter/simian virus 40 T-antigen fusion gene.

We have previously developed a transgenic (TG) mouse model expressing the Simian virus 40 T-antigen (Tag), driven by a 6-kb fragment of the mouse inhibin alpha-subunit promoter (inh-alpha). The mice develop metastasizing gonadal tumors, of granulosa/theca or Leydig cell origin, with 100% penetrance by the age of 5-8 months. In the present study, we examined whether the appearance and growth of the gonadal tumors are dependent on gonadotropins. Gonadotropin suppression was achieved either by treatment of 3-month-old mice for 2-3 months with a GnRH antagonist (Cetrorelix, SB-75), or by cross-breeding the TG mice to the genetic background of the gonadotropin-deficient hypogonadal mutant mouse (hpg). Gonadal tumor growth was clearly inhibited by SB-75 treatment in one of the TG mouse lines (IT6-M), as indicated by the absence of macroscopically visible tumors and by reduced gonadal weights. Despite the suppressed gonadotropin secretion and Tag expression, hyperplasia of testicular Leydig, and ovarian stromal cells persisted in some of the treated mice. In another TG mouse line (IT6-F), with more aggressive tumorigenesis, the SB-75 treatment only partially inhibited gonadal tumor growth. None of the hypogonadotropic TG mice, homozygous for the hpg mutation, developed gonadal tumors. Their gonadal histology was indistinguishable from that of the non-TG hpg mice, suggesting total inhibition of gonadal tumorigenesis in the absence of gonadotropin stimulation. Tag expression and Leydig cell hyperplasia were apparent already in the postnatal TG mice but absent in those TG mice homozygous for the hpg mutation. In conclusion, the present results indicate that the gonadal tumorigenesis in our TG mouse model starts in early age as hyperplasia in specific somatic cells. Both this, and the subsequent malignant tumor growth, are gonadotropin dependent. A sufficient level of Tag expression, a prerequisite for gonadal tumorigenesis, only occurs upon gonadotropin stimulation.

Pituitary and ovarian expression of the endogenous follicle-stimulating hormone (FSH) subunit genes and an FSH beta-subunit promoter-driven herpes simplex virus thymidine kinase gene in transgenic mice; specific partial ablation of FSH-producing cells by antiherpes treatment.

The ovarian expression of the endogenous follicle-stimulating hormone beta-subunit (FSH beta) and common alpha-subunit (C alpha) genes, and a herpes simplex virus thymidine kinase (tk) transgene, driven by a 2.3 kb bovine FSH beta promoter, was studied in normal and transgenic (tg) mice, tk functions not only as a neutral reporter that enables the study of the promoter function but also as an exogenously inducible toxigene. Reverse transcription-PCR followed by Southern blot hybridization with a nested probe was used to show the expression of the gene at the mRNA level. Common alpha-subunit mRNA was detected in the pituitary gland and ovaries of normal adult mice. We have previously detected endogenous FSH beta and tg tk mRNAs in the mouse pituitary, testis and ovary. In this study, the cellular localization of the corresponding proteins was visualized by immunocytochemistry. In normal mouse ovaries a positive reaction with FSH beta and C alpha antisera was seen in some of the corpora lutea and most prominently in the interstitial cells. A positive reaction with the tk antiserum was seen in the same cell types of tg mouse ovaries, but not in those of non-tg mice. Cell-ablation-inducing treatment (gancyclovir, 20 mg/kg per day, for 14 days) of tg female mice reduced pituitary FSH concentrations by 52% (P < 0.05) but did not affect pituitary LH or plasma gonadotropins compared with non-tg females treated in the same way. A longer period of cell ablation induction (acyclovir 400 mg/kg per day, for 21 days) reduced not only pituitary but also plasma FSH concentrations (55 and 57% respectively; P < 0.05) without affecting LH. This treatment also reduced ovarian weight by 38% (P < 0.01). In conclusion, our results show first that the endogenous FSH beta and C alpha proteins are produced in the mouse ovary. Hence, endogenously synthesized FSH or its subunits may have a role in the paracrine regulation of ovarian function. Secondly, the FSH beta promoter directs the expression of tg tk in the pituitary gonadotrope cells, as shown by specific but partial ablation of FSH-producing cells after induction by gancyclovir and acyclovir. In the ovary, tk protein was localized to the same compartments as the endogenous gonadotropin subunit proteins. This further confirms our finding of ovarian expression of the FSH subunit genes.

The FSH beta-subunit promoter directs the expression of Herpes simplex virus type 1 thymidine kinase to the testis of transgenic mice.

The bovine FSH beta-subunit promoter (2.3 kb) was coupled to the coding sequence of the Herpes simplex virus type 1 thymidine kinase (HSV-tk) gene and introduced into mouse embryos. A full-length tk transcript was found in the pituitary and testis. In the testis an additional truncated version of tk mRNA was also expressed. Two sets of primer extension fragments were identified, one corresponding to transcription initiation at or near the cap site of the FSH-beta gene, the other to transcription initiation within the tk gene. Furthermore, the latter, shorter transcript contained a 227 bp deletion. Only the long transcript was translated into immunoreactive tk in the later stages of developing spermatids. The tk protein was also functional in the testes, since spermatogenesis was either arrested or the germinal epithelium almost completely destroyed in transgenic males treated with the antiherpetic agent. If the FSH-beta-HSV-tk transgene also functions correspondingly in the pituitary, these mice will provide a useful model for studies on FSH.

Induced ablation of gonadotropins in transgenic mice expressing herpes simplex virus thymidine kinase under the FSH beta-subunit promoter.

Suppression of gonadotropins was induced by gancyclovir or acyclovir treatment in transgenic mice carrying 2.3 kb of bovine follicle-stimulating hormone beta (FSH beta) promoter fused to Herpes simplex virus thymidine kinase (tk) coding sequence. Transgenic tk and endogenous FSH beta were immunohistochemically co-localized in the same pituitary cells. In adult castrated transgenic males, gancyclovir treatment reduced plasma FSH (30%, P < 0.001). In intact juvenile gancyclovir treated mice, the reduction of pituitary FSH, and in males also of plasma FSH, was greater (50-70%, P < 0.05-0.01). A concomitant suppression of luteinizing hormone (LH) (50%, P < 0.01) was observed in female pups. The most pronounced reduction of gonadotropins was observed in newborn transgenic pups treated in utero with acyclovir. Both males and females had significantly lower pituitary levels of FSH (75-55%), LH (80-90%) or both (P < 0.05-0.01). Less pronounced decreases (30-40%, P < 0.01) were observed in plasma FSH. No apparent defects were seen in the testes of the transgenic, acyclovir treated, newborn pups. This mouse model is applied to study the dynamics of the gonadotropes and the role of gonadotropins in the maturation of the reproductive functions.

Transgenic mouse models for gonadal tumorigenesis.

The versatile transgenic (TG) techniques allow the production of in vivo animal models for a variety of diseases, including malignant tumors, through tissue-specific expression of oncogenes. We have created a TG mouse model for gonadal somatic cell tumors by expressing the powerful viral oncogene, Simian virus 40 T-antigen (Tag) under regulation of the murine inhibin alpha-subunit promoter (inh alpha). Ovarian granulosa and theca cell tumors were formed in the female, and those of testicular Leydig cells, in the male TG mice at the age of 5-6 months, with 100% penetrance. The tumors produced high levels of inhibin peptides, especially the alpha-subunit, and were steroidogenically active, mainly producing progesterone. The gonadal tumorigenesis was gonadotropin-dependent, since TG mice rendered gonadotropin-deficient by crossbreeding them into the hypogonadotropic hpg genetic background, or by treating them with a gonadotropin-releasing hormone (GnRH) antagonist, did not develop tumors. In order to study the possibility of using the tumor mouse model for testing gene therapy, we created another TG mouse model expressing under the same inhibin-alpha promoter the Herpes Simplex virus (HSV) thymidine kinase (TK) transgene. The inh alpha/HSV-TK mice were crossbred with the inh alpha/Tag mice and the double mutant mice also developed gonadal tumors. When they were treated with antiherpes drugs (acyclovir or gancyclovir), further growth of the tumors was blocked. These preliminary findings prove the principle that tumor ablation in our TG mouse model can be achieved by transduction of the HSV-TK gene into the tumor cells. Besides studies of formation, regulation and therapy of the tumors in vivo, immortalized cell lines derived from them provide models for studies of gonadal somatic cell functions in vitro.

The mouse inhibin alpha-subunit promoter directs SV40 T-antigen to Leydig cells in transgenic mice.

Testicular tumorigenesis was observed in transgenic mice expressing the 6-kb mouse inhibin alpha-subunit promoter/Simian virus 40 T-antigen (SV40 Tag) fusion gene. The tumors were confined to Leydig cells using immunohistochemistry with anti-Tag antibody, specific binding of biotinylated hCG and histochemistry for 3 beta-hydroxysteroid dehydrogenase. Leydig cell hyperplasia and presence of Tag protein in the testicular interstitial tissue were already evident at 5 and 6.5 days of age, respectively. An immortalized cell line, BLT-1, was established from one testicular tumor. These cells expressed the LH receptor and P450scc mRNAs, and displayed LH-responsive cAMP and progesterone production, and low testosterone production. The cells also specifically bound 125I-labeled recombinant human LH with high affinity (36000 binding sites/cell), and the binding was regulated by 8Br-cAMP and hCG. This gonadal tumor model is valuable for further studies on endocrine functions of Leydig cells and their tumorigenesis in vivo and in vitro.

Sex determination of bovine embryo blastomeres by fluorogenic probes.

One of the major challenges of using genetic information in marker assisted selection (MAS) is the detection of multiple marker loci from a small biopsy sample of a preimplantation stage embryo. The objective of this study was to develop a fast, nested, multiplex preamplification, polymerase chain reaction (PCR) method for the determination of sex in bovine embryo blastomeres. For this aim, ZFX/ZFY sequences were preamplified simultaneously with other genomic regions. The preamplification product was used as a template in an allelic discrimination assay, with nested primers and sex specific fluorogenic probes for ZFX and ZFY. Fluorogenic probes were used to eliminate the need for time consuming electrophoresis. Compared to sexing with Bovy/kappa-casein co-amplification method and other replicates from the same embryo, the accuracy of sexing with the use of fluorogenic probes after preamplification was 99% (112/113 blastomeres). The amplification efficiency was 96% (113/117 blastomeres).

Preimplantation genetic diagnosis in cattle: a review.

Preimplantation Genetic Diagnosis (PGD) is reviewed and novel fields where it may be applied are investigated. Technical advances of PGD in cattle embryos have already enabled its integration as a part of the MOET (Multiple Ovulation Embryo Transfer) breeding system. PGD for well-defined selection targets can enhance cattle breeding and embryo trade. It allows embryo selection according to their sex, and it may be used to breed special cow lines, or top bulls, by selecting embryos for valuable production traits using Marker Assisted Selection (MAS). A good allelic profile and/or the insertion of a transgene can be detected by PGD. This review article presents the technical requirements for PGD, and shows that this biotechnological method has great economic potential.

Transgenic animals and gonadotrophins.

A variety of transgenic animal models has been developed to elucidate in vivo the functions of gonadotrophin genes. Some of these have focused on regulatory aspects through expression of gonadotrophin subunit promoter-driven reporter genes. Others have been carried out by overexpression or targeted disruption of specific gonadotrophin subunit genes, or by eliminating pituitary gonadotroph cells in transgenic mice by expressing toxic transgenes under gonadotrophin subunit promoters. In addition, the overexpression or knock-out of genes of other hormones (for example GH and the inhibin subunits) has elucidated the regulation of gonadotrophin gene expression. Many of the transgenic animals produced serve as good models for human diseases affecting the hypothalamic-pituitary-gonadal function. This review summarizes the key results obtained with these novel genome modification techniques on the physiology and pathophysiology of gonadotrophin synthesis and secretion.

Apoptosis and cell proliferation potential of bovine embryos stimulated with insulin-like growth factor I during in vitro maturation and culture.

Reliable estimation and improvement of the developmental potential of in vitro production (IVP) embryos requires functional criteria of embryo quality. Antiapoptotic and mitogenic effects of insulin-like growth factor I (IGF-I), applied during bovine IVP, were studied. Day 6.5 blastocysts were fixed and processed for TUNEL to detect apoptotic cells, for immunocytochemical detection of proliferating cell nuclear antigen (PCNA), and for propidium iodide (PI) staining to detect all nuclei. Laser scanning confocal microscopy was used to determine apoptotic (TUNEL/PI) and proliferative (PCNA/PI) indices. Addition of IGF-I to the culture but not to the maturation medium increased the morula/blastocyst yield (P = 0.03), but the cleavage rate was not affected. During culture, IGF-I significantly lowered the apoptotic index by decreasing the number of apoptotic cells per embryo and elevated the total cell number of the blastocysts. The frequency of blastocysts with apoptotic cells was not affected. IGF-I increased the proportion of blastocysts with apoptotic cells in the inner cell mass area only by reducing apoptosis in the trophectoderm area. The PCNA index was not affected by IGF-I. A positive correlation observed between apoptotic and PCNA-positive cells was significant in groups stimulated with IGF-I during in vitro culture. Of TUNEL-positive cells, 30%-40% per embryo were also positive for PCNA. This colocalization may indirectly suggest an activation of DNA repair process in TUNEL-positive cells in response to DNA fragmentation. IGF-I reduces apoptosis in bovine IVP embryos. The requirement of IGF-I is more critical during embryo culture than during oocyte maturation. Our data suggest that an assay for TUNEL in conjunction with cell proliferation analysis can provide useful information about the quality of IVP embryos.

Systematic non-uniform distribution of parthenogenetic cells in adult mouse chimaeras.

Even though pure parthenogenetic mouse embryos die shortly after implantation, their cells are capable of participating in normal development of chimaeras when aggregated with fertilized embryos. Here we present data on parthenogenetic contribution to the oocyte populations measured by progeny tests in female chimaeras, and on distribution of parthenogenetic cells among the different organs by GPI typing. Systematic uneven distribution was detected. The highest level of participation was registered in the tissues of permanent cells (e.g. up to 63% in female germline). On the other hand, parthenogenetic cells were absent in several tissues that have extensive capacity for postnatal growth or selfrenewal. This finding suggests that uneven selective processes operate against parthenogenetic cells within certain differentiation pathways during fetal and postnatal life, as has already been observed in the development of extraembryonal membranes. It is likely that more than one mechanism is responsible for these selections. Parthenogenetic cells may start to differentiate in all cell lineages, but they are not able to react normally at certain points in the developmental pathway, for example to induction signals and, therefore, the cells fail to complete the normal processes of development, or to the proliferation requirement so that the fertilized counterpart gradually takes over the cell lineage. Paternally derived gene(s) might have a unique role in the development of tissues lacking parthenogenetic contribution.

Sex-chromosome linked gene expression in in-vitro produced bovine embryos.

The expression of XIST, G6PD, HPRT, ZFX and ZFY were investigated in in-vitro produced bovine embryos. Transcripts of these genes were assayed by RT-PCR in pools of pre-compaction stage embryos and sexed pools of morulae and blastocysts. The expression of XIST, G6PD, HPRT and ZFX in female and male morulae and blastocysts were compared using a semi-quantitative RT-PCR. G6PD, HPRT and ZFX transcripts were noted in all pre-compaction stage embryos and in female and male blastocysts. ZFY transcripts were detected in unsexed pools of 8-16-cell stage embryos and in male blastocysts. XIST transcripts were detected in unsexed pools at the 8-16 cell stage, in male and female morulae, and in female blastocysts. The level of XIST RNA was significantly higher in female morulae than in males. Levels of G6PD and HPRT RNA were also higher in female morulae and blastocysts than in males, but only G6PD levels were significantly different between the sexes. The expression of ZFX was also significantly higher in female than in male blastocysts. These results show sexually dimorphic expression of sex chromosome linked genes prior to the blastocyst stage in in-vitro produced bovine embryos.

Expression of the testis-specific HSP70-related gene (hst70 gene) in somatic non-testicular rat tissues revealed by RT-PCR and transgenic mice analysis.

The hst70 gene which belongs to rat HSP70 multigene family is highly expressed in pachytene spermatocytes. Using a transgenic mice model it was found that the promoter of the rat hst70 gene directs the expression of the chloramphenicol acetyl transferase (CAT) reporter gene not only to testis but also to multiple somatic tissues. In wild-type rats the expression of the hst70 gene in tissues other than testis was confirmed by non-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Beside the testis, the CAT expression in transgenic mice and the hst70 gene transcripts in wild-type rats were found in brain, pituitary, epididymis, vas deferens, adrenals, spleen, lung, ovary, oviduct and uterus. The only tissue in which both the CAT expression and the hst70 gene activity has not been found was the liver. These observations suggest possibly more universal, not confined to spermatogenesis, function of the hst70 gene.