The effect of relaxin on cell proliferation in mouse cervix requires estrogen receptor {alpha} binding to estrogen response elements in stromal cells.

The study objective was to determine whether stromal and/or epithelial estrogen receptor-alpha (ERalpha) is required for relaxin to promote proliferation of stromal and epithelial cells in the mouse cervix. Four types of tissue recombinants were prepared with cervical stroma (St) and epithelium (Ep) from wild-type (wt) and ERalpha knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of syngeneic female mice. After 3 wk of transplant growth, hosts were ovariectomized and fitted with silicon implants containing 17beta-estradiol (treatment d 1). Animals were injected sc with relaxin or vehicle PBS at 6-h intervals from 0600 h on d 8 through 0600 h on d 10. To evaluate cell proliferation, 5-bromo-2'-deoxyuridine was injected sc 10 h before tissue recombinants were collected at 1000 h on d 10. Relaxin promoted marked proliferation of both epithelial and stromal cells in tissue recombinants containing wt St (P < 0.001) but far lower proliferation in recombinants prepared with ko St, regardless of whether Ep was derived from wt or ko mice. An additional experiment using mice expressing wt ERalpha, a mutant of ERalpha that selectively lacks classical signaling through estrogen response element binding, or no ERalpha demonstrated that ERalpha must bind to an estrogen response element to enable relaxin's proliferative effects. In conclusion, this study shows that ERalpha-expressing cells in St, using a classical signaling pathway, are necessary for relaxin to promote marked proliferation in both stromal and epithelial cells of the mouse cervix.

Examination of the binding ability of bovine spermatozoa to the zona pellucida as an indicator of fertility.

Despite the development of many new techniques, laboratory assays still do not predict male fertility accurately. To identify targets for laboratory assessment, we first need to determine which steps in fertilization are most often defective in subfertile males. We developed a competitive in vitro fertilization assay in which spermatozoa from 2 different males, stained with different lipophilic dyes, are incubated together with oocytes in a droplet. By exposing mixed spermatozoa to the same oocytes, this assay controls for many of the variables of in vitro fertilization and should allow identification of the most common faulty steps in fertilization. The relationship of zona-binding ability to fertility is controversial. Therefore, as a first step, we determined if zona pellucida-binding ability, measured by this competitive assay, was related to bovine spermatozoal fertility. Fertility data were collected from 2 groups of bulls by 2 means of evaluation, nonreturn to estrus rates postinsemination and competitive insemination. In the nonreturn to estrus study, semen samples from 15 bulls were effectively ranked by zona-binding ability, using pairwise competitive in vitro zona-binding assays (R(2) = 0.84). However, this ranking was not significantly correlated with nonreturn rates (r = -0.04). In the competitive insemination study, semen samples from 8 bulls were effectively ranked by pairwise comparison using the competitive zona-binding assay (R(2) = 0.67). Again, this ranking was not significantly correlated to the competitive insemination index calculated for these bulls (r = 0.29). In the third study, we tested 3 bulls to determine if in vivo zona binding, assessed by the number of accessory spermatozoa, was correlated with in vitro zona binding. The number of accessory spermatozoa on oocytes recovered from cows after mating was not correlated with in vitro competitive binding of the spermatozoa. In conclusion, in vitro competitive zona binding was not correlated with bovine fertility or binding of accessory spermatozoa to oocytes in vivo.