The endometrium is receptive to embryo implantation only for a short period in each reproductive cycle: development of receptivity requires alterations in endometrial gene expression. Calbindin (CaBP)-d9k and CaBP-d28k are related proteins containing EF hand motifs that have a high affinity for Ca 2؉ . We previously demonstrated that endometrial expression of CaBP-d9k mRNA is highly regulated during implantation in the mouse. This project aimed to determine the temporal and spatial expression of both CaBP proteins during early pregnancy and to establish whether they are necessary for blastocyst implantation. CaBP-d28k protein, like CaBP-d9k, was up-regulated in the endometrial epithelium just before implantation but disappeared at implantation sites after attachment. By the judicious intrauterine injection of morpholino oligonucleotides (MO) against CaBP-d9k into WT and CaBP-d28k null mice just before implantation, we selectively eliminated one or both CaBPs from the uterine epithelium. Implantation was blocked only when both CaBP-d9k and CaBP-d28k were absent: treated WT mice and untreated CaBP-d28k null mice were fertile. Furthermore, the effect on implantation was highly dependent on the timing of injection of MO. This report examining the function of implantation-related genes in the uterus using MO demonstrates that this technique is a highly effective means to specifically target uterine proteins in vivo. This study provides evidence for an absolute requirement for CaBPs during the early phase of embryo implantation, and thus that regulation of Ca 2؉ availability in the uterine environment of the implanting embryo is critical for successful implantation.

The endometrium is hostile to embryo implantation except during the 'window of receptivity'. A change in endometrial gene expression is required for the development of receptivity. Calbindin-d9k (CaBP-d9k) and calbindin-d28k (CaBP-d28k) are proteins possessing EF-hand motifs which have high affinity for Ca 21 ions. Previously, it has been demonstrated that, in mouse endometrium, the expression of both calbindins is highly regulated during implantation and that both proteins play critical but functionally redundant roles at implantation. This study was the first to determine the expression of these two calbindins in the human and rhesus monkey endometrium. Initial RT-PCR analysis demonstrated that CaBP-d28k but not CaBP-d9k mRNA expression is detectable in the endometrium of both species. Western blot analysis confirmed the presence of immunoreactive CaBP-d28k protein in the primate endometrium. Furthermore, the endometrial expression pattern of CaBP-d28k mRNA and protein was examined by Northern blot analysis and immunohistochemistry respectively in both species across the menstrual cycle and during early pregnancy. Semi-quantitative statistical analysis of the immunohistochemistry results revealed that, in the human, CaBP-d28k protein expression was maximal in luminal and glandular epithelium during the mid-secretory phase, coinciding with the time when the endometrium is receptive to embryo implantation. Expression in rhesus monkey showed a similar trend. These results suggest that, in the primate endometrium, only CaBP-d28k is expressed and that the specific regulation of this calbindin is potentially important for the establishment of uterine receptivity.

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Calbindin-d9k (d9k) and calbindin-d28k (d28k) are highly upregulated in uterine epithelium at the time of implantation in mice 1,2 . This study aimed to investigate their functional roles in this process. Both wildtype (W/T) C57Bl6 and C57Bl6 tmpin mice (d28k-/-) were used, the latter strain can breed but with reduced fertility 3 . Uterine CaBP-d9k translation was disrupted using morpholino-modified anti-sense oligonucleotides (MO). Antisense d9k MO (30 nM/20µL in special delivery reagent EPEI) was administered by injection into one uterine horn day on either d2.5 or d3.5 prior to implantation (d4.5), (plug = d0). The other horn was injected with an irrelevant MO (n = 3/4 mice per treatment group). Numbers of implantation sites (N) were counted on d5.5. In preliminary studies intraluminal injection of FITC labelled MO showed their high integration into uterine luminal epithelium (the site of both d9k and d28k at implantation). Injection of specific MO into d28k-/-mice on d2.5 completely blocked implantation (Table 1), while no effect was seen following injection on d3.5. Similarly, no effect of MO injection was seen in W/T mice. The results show that endometrial expression of both d9k and d28k is necessary for implantation. The functions of these two as implantation can occur successfully when only one is present (i.e. in d28k-/-mice and in W/T treated with anti-d9k MO). The calbindins can now be added to the very small number of proteins shown to be critical for the process of implantation. These studies have clear implications for fertility regulation.(1) Nie et al. Biol. Reprod. 2000; (2) Luu et al. Proc. ASRB 2001; (3) Luu et al. Reprod. Fertil. Dev. Suppl. 14, 2002.