The Journal of Reproduction and Development Supplement

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Objective: Our previous studies have revealed that follicle stimulating hormone (FSH) inhibited apoptosis in pig granulosa cells. FSH stimulated hyaluronan (HA) synthesis. Furthermore the synthesized HA could decrease apoptosis, indicating that one of FSH inhibitory mechanism is mediated through HA. The present study was aimed to elucidate the apoptosis inhibitory action of HA in pig granulosa cell by analyzed the binding of HA and CD44, the receptor of HA. Materials and Methods: Pig follicles having 3-5 mm in diameter were isolated from ovaries. Cumulus-oocyte complexes with a granulosa layer which is designated as COCG, were dissected from follicles, and then cultured for 48 hours in DMEM/F-12 supplemented with FSH 50mU and various concentration of 4-MU (HA synthase inhibitor) or IM7 (anti-CD44 antibody) which reduced the binds of HA and CD44. The control group had only the medium. The protein expressions were detected using western blotting. We examined the expression of CD44 at COCG cell. Furthermore we analyzed the effect of HA-CD44 bind on apoptosis. Results: HA synthesis could be stimulated by FSH and inhibited by 4-MU. In this study, CD44 expressed after cultured for 48 h with FSH, decreased in addition of 0.1 mM/ml 4-MU, whereas no expression detected in high concentration of 4-MU, indicating that when the synthesis HA is inhibited, the amount of CD44 also decreased. Addition of IM7 decreased the expression of precursor caspase-3,8,9 indicating that the perturbation of HA-CD44 bind leads to activation of caspase-8,9 the initiator of apoptosis, also caspase-3 the executer of apoptosis. In conclusion, HA-CD44 interaction has been implicated in granulosa cells apoptosis inhibition.

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To study the roles of an anti-apoptotic factor, cellular FLICE-like protein (cFLIP), in the regulation of apoptosis in luteal cells of rat ovaries, we revealed that the changes in the expression levels and the localization of cFLIP mRNA and protein in CLs during estrus cycle and pregnancy. cFLIP short form and long form (cFLIPS and cFLIPL, respectively) mRNA and protein levels in the CLs which were categorized in first generation (G1) and second generation (G2) based on their structural and functional regressions and in those prepared from different pregnancy stages [1.0, 7.5, 9.0, 12.0, 13.5, 18.0 and 21.0 days of post coitus (dpc)] were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively, and that the changes in the localization of these proteins were demonstrated by immunohistochemistry. Lower levels of cFLIPS and cFLIPL mRNAs were noted in CLs with proestrus stage of G2, and higher levels were noted in CLs of mid pregnancy stages (12.0 and 13.5 dpc). Higher expression levels of cFLIPS protein were seen in CLs of mid of pregnancy stages and lower levels in CLs with proestrus stage of G2. Strong positive stainings for cFLIPS/L proteins were demonstrated in CLs of mid of pregnancy stages. In the present study, we firstly showed the changes in cFLIP expression levels and the localization in CLs during estrus cycle and pregnancy in rat, and these findings suggest that cFLIP may acts as a dominant survival factor by inhibiting intracellular apoptosis signal transduction in rat CLs.

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The objective of this study was to investigate the effect of post-AI luteal function on first AI conception rate in dairy cows. A total of 119 Holstein Friesian cows from four dairy herds were used in this study. Milk samples were collected twice a week and progesterone concentrations in whole milk were measured by direct ELISA. The luteal function post-AI was shown by days to commencement of luteal activity after AI, progesterone concentrations during early luteal phase and these combinations. Cows with commencement of luteal activity during a period from 1 or 2 to 5d post-AI had the highest conception rate. Increasing progesterone concentrations during 5 to 9d post-AI were associated with higher conception rates. Animals with delayed luteal activity 9d post-AI or later, those with delayed and insufficient luteal activity and those with insufficient luteal activity had lower conception rates compared to cows with normal luteal activity (22.2%, 12.5%, 0.0%, vs 51.5%, respectively; P<0.10). Logistic regression analysis revealed that CL function (OR=0.6; p=0.001) significantly affects the first AI conception rate, whereas the other factors including parity, resumption of ovarian activity postpartum, season of AI and interval from calving to first insemination had no significant effect. In conclusion, both commencement of luteal activity post-AI and progesterone concentration during early luteal phase affect the conception rate.

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A new progesterone intra-vaginal device for cattle has been developed in Argentina. The product (DIB) is Y-shaped and contains 1 g progesterone. The objective of this study was to know the effectiveness of DIB, in comparison with CIDR, in estrus synchronization of lactating dairy cows. A total of 110 Holstein Friesian cows from 22 commercial dairy farms in Yamaguchi Prefecture were used. The cows were 60d postpartum or later and were in their first to fourth lactations, and had no clinical abnormality in ovaries and uterus. The 110 cows were divided into two treated groups. One group of 54 cows was inserted into the vagina with DIB, which was removed 12d after insertion. The other group of 56 cows were treated with CIDR as same as DIB treated group. Milk samples were collected at 0, 7, 12, 14 and 21d after insertion to measure progesterone concentrations. Decrease in milk progesterone concentrations 14d after insertion (2d after removal) was considered to be positive response. Estrus was detected by visual observation by farmers with or without heat detection aids. Cows were inseminated artificially after estrus detection. Estrus detection rate within 6d after removal of CIDR and DIB was 57.1% and 44.4%, respectively. Estrus induction rate based on milk progesterone profile and expression of estrus was 64.3% and 66.7% in CIDR and DIB groups, respectively. Conception rate after AI at induced estrus was 21% in CIDR group and 19% in DIB group. It is concluded that DIB is equally effective to CIDR in estrus synchronization in dairy cows.

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