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Important work but with some questions

Posted by allen1 on 07 Mar 2009 at 11:39 GMT

I think it is a important work which introduced RNAi technology into the study of male reproductive tract. However, several points should be addressed.

(1) It is a pity that the authors have not performed the rescue experiment. Because RNAi usually has an off-target effect, although authors used two different RNAi fragments, it is still better to add HongrES1 to restore the phenotype, such as sperm-HongrES1 protein incubation in vitro to demonstrate HongrES1 is a decapacitation factor.

(2) In RNAi experiment, authors used a non-silencing siRNA (Csi) from one company (Shanghai GeneChem Inc)as negative control. As we know, RNAi and miRNA both use RISC complex. I do not know Csi in this paper can be incorporated into RISC. If not, this control is not very strict because in this case Csi will not affect miRNA function by occupying RISC but the two RNAi fragments for HongrES1 might affect miRNA pathway.

(3) It is really interesting the antisera against HongrES1 can induce capacitation. It is a pity that authors seemed not to put related procedure in materials& methods. Did authors use purified antisera or unpurified antisera? What is the antisera concentration during the incubation? The authors also did not show the amount change of HongrES1 on sperm after sperm-antisera incubation.

(4)The sperm is very sensitive and might be impaired during the in vivo electroporation experiment. The authors did not show the immunohistochemistry result after RNAi injection otherwise we can know siRNA can be spread into the exact region in cauda epididymis.

(5) In figure 3 authors show the result of 5hr sperm incubation for capacitation but in figure 4 authors only show the result of 3hr sperm incubation. How about the result of 4-5hrs incubation ?

(6) It is surprising that authors can get at least 69 successful mating in 48-60 hrs after cut-suture experiment around the scrotum with only one night male-female incubation (Figure 5B). Usually the animals will lose any mating interest because of the pain and smell from the wound during this short period(48hr-60hr). I do not know how many animals authors used to get so many successful matings.

RE: Important work but with some questions

zhouych replied to allen1 on 18 Mar 2009 at 06:26 GMT

In reply to the comments of Allen Wu,
(1) That’s a good point. In fact, we tried purifying the protein with mammalian express system to get active protein, but failed. Actually, we pointed out the situation in the discussion (active HongrES1 recombinant protein expression will be a key step in further studies). Recently, our collaborators have demonstrated that the HongrES1 protein can inhibit the capacitation of sperm (unpublished data). We hope we can tell you more latterly.
(2) The control fragments were designed by following the common rules, i.e. the same length and GC percentage as the test fragments, and not match any gene by blast to the whole genome. We think that’s a good control.
(3) The raw anti-serum was tittered by the bacterial expressed antigen and verified by western blot analysis. For the experiments, the samples were incubated with raw anti-serum for 1 to 5 hr in the concentration of 1:250.
Checking the HongrES1 level after sperm-antiserum incubation is another good point, we missed this point.
(4) Sorry, we did not perform these observations.
(5) We described the details of the experiments in the text. Obviously, both in vivo and in vitro tests have significant difference. For the in vivo experiment, we did not observe the 5hr time point when the sperm mobility decreased dramatically. We discussed this issue with a Germany colleague, and he told us it is very difficult to check the capacitation state if the motility of sperm was reduced dramatically.
(6) We worry about the situation before the experiment too. To confirm that the male rats with scrotum surgery do mate the female rats well, we did lots of preliminary tests, and got successful mating. For the experiment, we performed the surgery very carefully to reduce the damage to the animals. Since the HongrES1 was mostly expressed in the cauda of epididymis, we cut a small incision on the scrotum, and then gently push the epididymis cauda out to inject the RNAi fragment. In addition, we used sterile PBS instead of 70% ethanol to reduce the pain during the operation. Moreover, we tested no more than six male rats (Csi: 1 or 2; Hsi1: 1 or 2; Hsi2: 1 or 2) every time to keep control for high efficiency.
As for the amount of the test rats, we successfully performed RNAi injection on 156 healthy male rats, then mated them with 156 healthy females separately. Among the mated females, 69 were pregnant.