Category Archives: Ribonucleic Acid

Characterization of Inhibin/Activin Subunit: DISCUSSION(5)

In comparison to signals obtained from oocytes, we detected relatively low levels of both ZP3 and GDF9 mRNAs in human cumulus cells. While both ZP3 and GDF9 appear to be oocyte specific in mice, several investigators have observed ZP3 mRNA and/or protein in granulosa cells from other species, including humans. Thus, ZP3 mRNA detection in human granulosa-lutein cells is consistent with these earlier observations and demonstrates that human granulosa cells, like those from pigs and rabbits, can synthesize ZP3. buy yasmin online

Characterization of Inhibin/Activin Subunit: DISCUSSION(4)

The recent immunocytochemical identification of type II activin receptors on mouse oocytes further supports this concept. buy flovent inhaler

In our experiments, the PCR product from activin receptors appeared to vary significantly across oocyte meiotic maturational stages. ActRIIB and ActRIB mRNA levels were significantly lower at Ml compared to either GV or M2. ActRIIA mRNA, on the other hand, was lower at Ml but remained reduced at M2. To examine these potentially quantitative relationships more closely, we repeated these experiments on a smaller group of human oocytes using a semiquantitative RT-PCR protocol with amplification in the exponential range for each target.

Characterization of Inhibin/Activin Subunit: DISCUSSION(3)

To ensure that only oocyte mRNA was analyzed in our study, oocyte RNA samples were examined for the ovarian form of aromatase, and all samples were deleted in which significant aromatase was detected. Thus, differences between our results and those in previous reports of activin subunit mRNA in oocytes using RT-PCR may be accounted for by residual granulosa cells adhering to oocytes at the time of extraction, since the oocytes were not individually examined before extraction and ovarian control PCR reactions were not performed. Furthermore, our failure to detect inhibin/activin subunit mRNA in oocytes is consistent with previous in situ hybridization studies.

Characterization of Inhibin/Activin Subunit: DISCUSSION(2)

Interestingly, the only difference between mouse and human oocytes in this analysis was the presence of FS mRNA in human but not mouse oocytes. The mouse primers were located identically to the human primers, and the FS gene is highly conserved between species, so this difference is not likely due to differences in PCR sensitivity. Moreover, FS mRNA was detected in mouse embryos as well as in adult ovaries, both of which served as positive controls. FS has been previously demonstrated to bind activin nearly irreversibly and to neutralize activin’s bioactivity. Thus, if the FS mRNA in human oocytes is translated, this difference in FS expression might make human oocytes more resistant to the effects of activin relative to mouse oocytes, or limit the time over which activin can exert its signal. birth control yasmin

Characterization of Inhibin/Activin Subunit: DISCUSSION(1)

To further examine the hypothesis that activin is an in-trafollicular paracrine signal between granulosa cells and oocytes, we examined mRNA levels of the inhibin/activin subunits, FS315, and activin receptors in individual human oocytes to determine which of these molecules are synthesized. While mRNAs for each of the four activin receptor subtypes were easily detected, we found no significant mRNA for any of the inhibin/activin subunits, suggesting that neither inhibin nor activin is produced by oocytes.

Characterization of Inhibin/Activin Subunit: RESULTS(2)

Surprisingly, low levels of GDF9 and ZP3 mRNA, which were included as ‘‘oocyte-specific’’ markers, were observed in 40% and 80% (respectively) of the cumulus samples. antibiotics levaquin

Expression of activin receptor subtype and FS315 mRNA was examined during oocyte meiotic maturation. All mRNA species examined were detectable at all three nuclear maturational stages. However, the steady-state mRNA levels for ActRIIA, ActRIIB, and ActRIB appeared to vary during maturation. In particular, the ActRIIB PCR product signal intensity was lower (p < 0.05) in M1 oocytes compared to either GV or M2 oocytes, while for ActRIB, the PCR signal intensity was lower in M1 oocytes than in M2 oocytes (p < 0.05). For ActRIIA, the PCR product in GV oocytes appeared to be higher than that in either M1 or M2 oocytes (p = 0.05 and p < 0.05, respectively).

Characterization of Inhibin/Activin Subunit: RESULTS(1)

The PCR product for each target from individual human oocytes was distinguishable from that of cumulus cells, both in absolute amount and in the fraction of samples expressing each target (Table 2), the latter being an indication of the relative abundance of an individual transcript. Specifically, neither (3A- nor (3B-subunit mRNA was detectable in any oocyte, while low levels of a-subunit mRNA were detected in only 11% of human oocytes. FS315 mRNA was detected in 95% of human oocytes, but the signal intensity was relatively low compared to that in cumulus cells. On the other hand, robust signals were detected for activin receptor subtype transcripts in nearly all oocytes (88-100%). ampicillin antibiotic

Characterization of Inhibin/Activin Subunit: MATERIALS AND METHODS(5)

In addition, no specific bands were detected in reactions containing the tRNA carrier alone. To confirm the integrity of the RNA templates and the RT-PCR protocol, p-actin was examined in all samples. Furthermore, zona pellucida 3 (ZP3) and GDF9 were amplified as oocyte-specific markers, while the ovary-specific transcript of the human P450arom was used to ensure the absence of granulosa cell mRNA in the oocyte RNA extracts. For transcripts that were negative in human oocytes, granulosa cell RNA was used as the positive control, while mouse ovary total RNA was used as a positive control sample for mouse oocytes. Mouse embryos were analyzed as a control for the sensitivity of the PCR for targets that were undetectable in oocytes. Buy Advair Diskus Online

Characterization of Inhibin/Activin Subunit: MATERIALS AND METHODS(4)

Most of the primers used in this study for human or mouse FS, a, pA, and pB inhibin/activin subunits, the four activin receptor subtypes, and p-actin were previously described and demonstrated to provide single, specific PCR bands whose identities were confirmed by Southern blotting or direct sequencing. The sequences of primers not previously utilized are listed in Table 1, and their PCR products’ identities were verified by subcloning (TA cloning kit; Invitrogen, San Diego, CA) and sequencing. A cDNA fragment of human growth-differentiation factor-9 (GDF9; ) was cloned and sequenced by amplifying human oocyte cDNA with the mouse primers. The human-specific primer set (Table 1) was positioned within this fragment.

Characterization of Inhibin/Activin Subunit: MATERIALS AND METHODS(3)

Complementary DNA-bound RNA was then removed by RNAse H (2 U; GibcoBRL) digestion at 37°C for 20 min. PCR was performed in a 25-^l reaction containing an RT aliquot equivalent to approximately 10% of the RT reaction, 0.2 mM dNTP mix, 0.25-0.5 ^M upstream and downstream primers (each), 0.5 ^Ci [a-32P]CTP (3000 Ci/mmol; New England Nuclear, Boston, MA), and 1.25 U Taq polymerase in single-strength PCR buffer A (both from Fisher Scientific, Pittsburgh, PA). After denaturation at 94°C for 3 min, templates were amplified for 22 cycles using a temperature profile of 94°C, 58°C, and 72°C for 30 sec each in GeneAmp PCR System 9600 (Perkin Elmer, Norwalk, CT). my canadian pharamacy

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