Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium.

BACKGROUND: Various molecules participate in implantation and maintaining endometrial function during gestation. The remodeling of endometrial matrices is a necessary process in the coordination of gestational progress. Matrix-metalloproteinases (MMPs) like gelatinases (MMP-2 and -9) and collagenase (MMP-1) are considered to play important roles in this process. We examined MMP-2 and -9 expression using zymography, in situ hybridization, real-time PCR, and microarray analysis to clarify their roles in the bovine endometrium during gestation. METHODS: Endometria, placentomes, and fetal membranes were collected from Japanese black cows that were killed on day 15 to 252 of gestation or during their estrous cycle. The gene expression of MMP-related molecules (mainly MMP-2 and -9) was examined using a custom-made microarray, real-time RT-PCR, and in-situ hybridization. Gelatinase activity was detected by zymography and film in situ zymography. RESULTS: Both gelatinases were expressed in the endometrium and fetal tissues throughout gestation. MMP-2 gene expression declined with the progress of gestation, but its intensity was maintained at a high level during the peri-implantation period and increased in late gestation. The expression level of MMP-9 was stably maintained, but was relatively low compared to that of MMP-2. These gene expression patterns matched those detected by zymography for the proteins. Microarray analysis suggested that the functions of MMP-2 during implantation and the last part of gestation are closely related with those of other molecules such as tissue inhibitors of metalloproteinase (TIMP)-2, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 1, membrane type 1 (MT1)-MMP, and extracellular matrix metalloproteinase inducer (EMMPRIN). CONCLUSION: We detected MMP-2 and -9 gene expression in the bovine endometrium and placentome throughout gestation. These data suggest that MMP-2 is one of the main endometrial remodeling factors for implantation and pre-partum in cattle. In cows, as is the case in humans and rodents, gelatinases participate in endometrial remodeling, and their activities depend on the balance of activators and inhibitors; i.e., TIMP, MT-MMP, EMMPRIN, MMP-2, MMP-9, and so on.

Formation of follicular cysts in cattle and therapeutic effects of controlled internal drug release.

Follicular cysts in cattle result from excessive growth of the dominant follicle without ovulation and still constitute a major reproductive disorder in this species. One key hormonal characteristic of cows with follicular cysts is the lack of an LH surge, although they have increased plasma estradiol concentrations. Another is a relatively high level of pulsatile secretion of LH that promotes continued growth of the dominant follicle. These LH characteristics seem to result from a functional abnormality in the feedback regulation of LH secretion by estradiol. Treatment with controlled internal drug release devices that increase circulating progesterone levels is effective in resolving follicular cystic conditions by 1) lowering pulsatile LH secretion and 2) restoring the ability of the hypothalamo-pituitary axis to generate an LH surge in response to an increase in circulating estradiol.

Plasma concentrations of inhibin A in cattle with follicular cysts: relationships with turnover of follicular waves and plasma levels of gonadotropins and steroid hormones.

We investigated the profiles of circulating levels of inhibin A and total inhibin in beef cows with follicular cysts in relation to the patterns of follicular development and circulating gonadotropins and steroid hormones. Turnover of follicular waves was monitored in five cows every 2 days for 70 days from 10 days after detection of estrus without ovulation. The mean interwave intervals were 19.6 +/- 1.0 days (n = 18 waves with cysts from the five cows). Circulating levels of inhibin A were approximately 170 pg/ml before emergence of follicular waves with cysts and increased (P < 0.05) concomitantly with follicle emergence. High concentrations of inhibin A (greater than 300 pg/ml) were noted for 7 days during the growth phase of cystic follicles, but inhibin A levels decreased gradually when development of the cysts reached a plateau. This profile of inhibin A was similar to those of total inhibin and estradiol, but was inversely related to the changes in plasma FSH concentrations. LH pulse frequency and mean concentrations of LH in cows with cysts were higher than those observed in the luteal phase of normal cyclic cows. These results indicate that the capacity to secrete inhibin, as well as estradiol, is maintained in cystic follicles, the growth of which is extended by LH secretion at levels greater than those seen in the normal luteal phase. Inhibin A plays an important role in the extension of interwave intervals by suppressing recruitment of a new cohort of follicles.

Retrospective analysis of the efficacy of controlled internal drug release in follicular cysts in an embryo donor beef herd.

We investigated the efficacy of a controlled internal drug release (CIDR) device in the reproductive management of an embryo donor beef herd. Superovulation of embryo donors was routinely induced by a combination of FSH and prostaglandin F(2)alpha analogue (PGF(2)alpha) at intervals of a few months, and after embryo recovery estrus of the donors was synchronized with PGF2(2)alpha. Between 1996 and 1998, approximately 20% of donors were diagnosed as having follicular cysts every year. Twenty-eight cases of follicular cysts recorded between 1997 and 1998 were treated with a CIDR device for 14 days to evaluate the efficacy of CIDR in resolving follicular cysts in donor herds. Initial recovery was defined as the occurrence of ovulation with estrous behavior and subsequent formation of a corpus luteum after removal of the CIDR. Initial recovery was recognized in all cases (n=28). Follicular cysts did not recur after repeated embryo recovery in 64% of the initially recovered donors, but in 36% of affected donors cysts recurred after the next embryo recovery. Subsequently, with a CIDR device instead of PGF(2)alpha, we synchronized estrus after embryo recovery in the same herd from 1999 to 2000, to investigate the ability of CIDR to prevent the initiation of follicular cysts. Of the donors used between 1999 and 2000, approximately 30% had a history of follicular cysts. Use of CIDR for estrous synchronization after embryo recovery lowered the incidence of follicular cysts to 3% in 1999 and 0% in 2000. Treatment with CIDR proved effective at resolving follicular cysts in the embryo donor beef herd and enabled re-use of donors affected with follicular cysts. CIDR is also likely to be efficacious in lowering the occurrence of follicular cysts in donor herds when it is used for estrous synchronization after embryo recovery.

Characterization of gene expression profiles in early bovine pregnancy using a custom cDNA microarray.

Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placental-specific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy.

Matrix-metalloproteinases-2 and -9 production in bovine endometrial cell culture.

In vitro cell culture is a convenient tool for studying cellular mechanisms. In the present study, production of matrix-metalloproteinases (MMPs) in bovine endometrial (containing both epithelial and stromal cells) monolayer cells was examined. Blastocysts attached to the endometrial cells in a monolayer culture were examined for their effects on MMP-2 production. Initial attachment of blastocysts to the monolayer inhibited MMP-2 production by endometrial cells. But once trophoblast cells began to migrate into the endometrial cell layer, MMP-2 production increased, and at the same time MMP-9 production also became evident in the medium. In order to understand how blastocysts affected MMP-2 production, we examined the effect of progesterone, estradiol, insulin-like growth factors (IGFs), tumor necrosis factors (TNFs), and interferon-tau (IFN-tau) supplementation. It was IFN-tau that inhibited the production of MMP-2. In addition, progesterone at a lower dose appeared to inhibit MMP-2 production. Both TNF-alpha and TNF-beta strongly stimulated the production of MMP-2 and MMP-9, whereas IGFs had no effect. Based on these findings, it appears that conceptus has the capacity to inhibit MMP activity.

Implantation and placental development in somatic cell clone recipient cows.

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.

The dynamic expression of extracellular matrix in the bovine endometrium at implantation.

Remodeling of uterine endometrial extracellular matrix (ECM) is pivotal to successful implantation and placentation, and has been well described in the rodents and humans. However, bovine endometrial ECM remodeling is still vaguely defined, especially at the time of implantation. Therefore, this study investigated the distribution of four ECMs namely, types I and IV collagen, laminin and fibronectin, from days 0 to 30 of gestation in bovine endometrium by immunofluorescence microscopy. A change in the distribution pattern of ECMs was evident by day 14 of gestation as features at this stage were clearly different from those of day 14 of the estrous cycle. The immunoreactivity of type I collagen, fibronectin and laminin decreased from day 14 of gestation and was obscured by day 24 of gestation. The type I collagen fibers formed were of thinner consistency than those of the estrous cycle and showed a coarser meshwork within the epithelium sites during the implantation period. In addition, the type IV collagen and laminin immunoreactivities of epithelial basement membrane also remarkably declined at exactly the same time. By day 30 of gestation, the four ECMs had regenerated with the formation of the placentome. In conclusion, this study reveals that remodeling of ECM is essential for the successful establishment of pregnancy in the bovine.