hi rosie, I have not seen this in a paper. usually people use serum gonadotropins and/or vaginal smears, after all these could be done by someone else or someone else could give the samples a different coded name so you still don't know until decoded which samples go with which brains.

but assuming you are asking because you have the brains but do not have these samples, I wonder if you could look at the levels of GnRH and GnRH receptor messenger or better yet a ratio of the two. (see Schirman-Hildesheim TD, Bar T, Ben-Aroya N, Koch Y. Endocrinology. 2005 Aug;146(8):3401-8.)

unless someone knows if this has been successfully done before, it would need to be validated, so you would need those samples anyway but I wonder if anyone reading has an opinion/experience?

note, probably the best, most sensitive test is behaviour with a sexually experienced male rat

hi rosie, I have not seen this in a paper. usually people use serum gonadotropins and/or vaginal smears, after all these could be done by someone else or someone else could give the samples a different coded name so you still don't know until decoded which samples go with which brains.

but assuming you are asking because you have the brains but do not have these samples, I wonder if you could look at the levels of GnRH and GnRH receptor messenger or better yet a ratio of the two. (see Schirman-Hildesheim TD, Bar T, Ben-Aroya N, Koch Y. Endocrinology. 2005 Aug;146(8):3401-8.)

unless someone knows if this has been successfully done before, it would need to be validated, so you would need those samples anyway but I wonder if anyone reading has an opinion/experience?

note, probably the best, most sensitive test is behaviour with a sexually experienced male rat

Similar to what Pasha and Vicky suggested, you can look at kisspeptin or it's receptors expression in the hypothalamus. (A good starting point would be this paper http://endo.endojournals.org/content/147/6/2864.full).

I'd suggest looking at GnRH, kisspeptin and GnIH expression, after doing a lit review to see if someone has charted the changing expression in the rat.

I would just like to stress that, as Vicky said, all of the methods mentioned here would need to be validated and, especially if doing this in mice, I would be extremely careful in using gene expression to determine estrous cycle stage. You may possibly get away detecting proestrous, if animals were sacrificed in the afternoon, but I doubt you will reliably distinguish, the other stages.

The best and easiest thing to do would be to get vaginal smears from the next lot of animals you use for your experiments. From my experience, I would say this is way more reliable (not to mention much easier!) than measuring serum gonadotropines/hormones.

Hello all!

Thanks so much for your interesting responses. As some of you queried, I do only have brain tissue (and some bladder, but I'm guessing that wouldn't be so useful) from perfused animals, which as it happens is already stained for cFos! Thanks o much for the offer of primer Pascha - I was actually doing some PCR today, but on something else :p Would be great to look at that but I'd need more tissue.

Nicola - I was pretty consistent with my perfusion timings (11-3pm) so if I had fresh tissue, that would have been perfect.

I opted against ovariectomizing my rats as I'm looking at complex behaviour so wanted to minimise surgery - and as I mentioned felt that as in the clinic (or in trials AFAIK), women are rarely tested for menstrual stage, it was worth seeing if effects could be detected in a mixed population. I did see effects, and so maintained this protocol, but was just curious about whether any retrospective tests existed! I'm almost at the end of my PhD, so it's unlikely I'll be running any more batches, however if I have time, I will definitely look at cFos in the AVPV - as much as assumptions are dangerous, would I expect to see a similar profile within batches (i.e. animals that all arrived on the same date)?

Thanks again for all your answers - if I see anything of interest, I'll be sure to report back :)

Recent studies have shown that the concentration of 17β-estradiol, determined by mass spectrometry, in the rat hippocampus is similar in adult tissues obtained from rats at proestrus and estrus. The concentration of 17β-estradiol in the hippocampus in both stages of the estrous cycle is higher than the plasma concentration, indicating local synthesis hormone. Furthermore, it has been reported that the effects of exogenous 17β-estradiol administration depend of the difference in the concentration between the exogenous administered 17β-estradiol and the level of endogenous 17β-estradiol.

References:

1- Hojo Y, Higo S, Ishii H, Ooishi Y, Mukai H, Murakami G, Kominami T, Kimoto T, Honma S, Poirier D, Kawato S. Comparison between hippocampus-synthesized and circulation-derived sex steroids in the hippocampus. Endocrinology. 2009; 150: 5106-12.

2- Mukai H, Kimoto T, Hojo Y, Kawato S, Murakami G, Higo S, Hatanaka Y, Ogiue-Ikeda M Modulation of synaptic plasticity by brain estrogen in the hippocampus. Biochim Biophys Acta. 2010; 1800: 1030-44.