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.

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.

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.

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