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      Pattern of gonadotropin secretion along the estrous cycle of C57BL/6 female mice

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          Abstract

          The pattern of gonadotropin secretion along the estrous cycle was elegantly described in rats. Less information exists about the pattern of gonadotropin secretion in gonad‐intact mice, particularly regarding the follicle‐stimulating hormone (FSH). Using serial blood collections from the tail‐tip of gonad‐intact C57BL/6 mice on the first day of cornification (transition from diestrus to estrus; hereafter called proestrus), we observed that the luteinizing hormone (LH) and FSH surge cannot be consistently detected since only one out of eight females (12%) showed increased LH levels. In contrast, a high percentage of mice (15 out of 21 animals; 71%) exhibited LH and FSH surges on the proestrus when a single serum sample was collected. Mice that exhibited LH and FSH surges on the proestrus showed c‐Fos expression in gonadotropin‐releasing hormone‐ (GnRH; 83.4% of co‐localization) and kisspeptin‐expressing neurons (42.3% of co‐localization) of the anteroventral periventricular nucleus (AVPV). Noteworthy, mice perfused on proestrus, but that failed to exhibit LH surge, showed a smaller, but significant expression of c‐Fos in GnRH (32.7%) and AVPV Kisspeptin (14.0%) neurons. Finally, 96 serial blood samples were collected hourly in eight regular cycling C57BL/6 females to describe the pattern of LH and FSH secretion along the estrous cycle. Small elevations in LH and FSH levels were detected at the time expected for the LH surge. In summary, the present study improves our understanding of the pattern of gonadotropin secretion and the activation of central components of the hypothalamic–pituitary‐gonadal axis along the estrous cycle of C57BL/6 female mice.

          Abstract

          Our study exposed several challenges in working with gonad‐intact mice to evaluate the pattern of the HPG axis, especially during the LH surge. We observed that LH surge is often not observed in regular cycling C75BL/6 mice. We found evidence that in some cases in which a full LH surge is not observed, the HPG axis is somewhat activated, either through a small percentage of c‐Fos expression in MPO GnRH and AVPV Kisspeptin neurons or via a small peak in LH levels.

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          Mouse Estrous Cycle Identification Tool and Images

          The efficiency of producing timed pregnant or pseudopregnant mice can be increased by identifying those in proestrus or estrus. Visual observation of the vagina is the quickest method, requires no special equipment, and is best used when only proestrus or estrus stages need to be identified. Strain to strain differences, especially in coat color can make it difficult to determine the stage of the estrous cycle accurately by visual observation. Presented here are a series of images of the vaginal opening at each stage of the estrous cycle for 3 mouse strains of different coat colors: black (C57BL/6J), agouti (CByB6F1/J) and albino (BALB/cByJ). When all 4 stages (proestrus, estrus, metestrus, and diestrus) need to be identified, vaginal cytology is regarded as the most accurate method. An identification tool is presented to aid the user in determining the stage of estrous when using vaginal cytology. These images and descriptions are an excellent resource for learning how to determine the stage of the estrous cycle by visual observation or vaginal cytology.
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            Kisspeptin-GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge.

            Kisspeptin and its receptor GPR54 have recently been identified as key signaling partners in the neural control of fertility in animal models and humans. The gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons of the neural network controlling fertility and are suspected to be the primary locus of kisspeptin-GPR54 signaling. Using mouse models, the present study addressed whether kisspeptin and GPR54 have a key role in the activation of GnRH neurons to generate the luteinizing hormone (LH) surge responsible for ovulation. Dual-label immunocytochemistry experiments showed that 40-60% of kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) expressed estrogen receptor alpha and progesterone receptors. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, approximately 30% of RP3V kisspeptin neurons were found to express c-FOS in surging mice compared with 0% in nonsurging controls. A strong correlation was found between the percentage of c-FOS-positive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 were essential for GnRH neuron activation and the LH surge, Gpr54- and Kiss1-null mice were examined. Whereas wild-type littermates all exhibited LH surges and c-FOS in approximately 50% of their GnRH neurons, none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin-GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. This broadens considerably the potential roles and therapeutic possibilities for kisspeptin and GPR54 in fertility regulation.
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              The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy.

              The hormonal factors associated with converting a corpus luteum of estrous cycle into a corpus luteum of pseudopregnancy were studied by measuring LH and FSH prolactin, estradiol and progesterone levels in decapitated rats during the 4-day estrous cycle and a comparable time of pseudopregnancy (lights on 0600-0800 hr.). During the estrous cycle, prolactin, LH and FSH remained low and unchanging except on the afternoon of proestrus, when typical proestrous surges were observed. In contrast, estradiol levels began to increase on D-1, from baseline values of 7 pg/ml to approximately 15-20 pg/ml. These levels were maintained until the afternoon of D-2 when estradiol further increased to reach peak levels of 40-50 pg/ml by 0900 hr on proestrus. Estradiol then declined in relation to the increase in LH secreation and had returned to baseline by estrus. Progesterone secretion by the corpora lutea of the cycle also increased on the afternoon of D-1 and reached a maximum value of 25-30 ng/ml early on the morning of D-2. At this time, a precipitious fall in progesterone occurred, returning to baseline values of 5-1- ng/ml by 0700 on D-2 signifying the regression of the corpora lutea of the cycle. Progesterone remained low thereafter until the afternoon of proestrus when levels increased in response to the proestrus when levels increased in response to the proestrous surge of LH. Following cervical stimulation at 1900 hr on proestrus, no differences were noted, with respect to the estrous cycle, in LH, FSH or estradiol secreation through the afternoon of D-2. Surprisingly, progesterone levels did not differ in the cycle and pseudopregnancy until the early morning of D-29 instead of progesterone levels falling to baseline as they had during the cycle, the corpora lutea of pseudopregnancy were rescused, progesterone increasing dramatically to reach levels of 45-50 ng/ml by 1700 hr on that same day. The only difference in hormone secretion that was noted which could account for this marked divergence in progesterone secretion was the pattern of prolactin secretion following cervical stimulation. In contrast to the low levels seen during the estrous cycle, biphasio surges of prolactin secretion occured each day, one being nocturnal (0100-0900 hr) and the other diurnal (1500-2100 hr). The rescue of the corpus luteum occured in association with the nocturnal surge on D-2. These results suggest that nocturnal surge on D-2, PROLACTIN IS THE MAJOR Luteotropic stimulus which transforms and estrous cycle into pseudopregnancy by prolonging progesterone secretion from the corpus luteum. Moreover, if LH is important for progesterone secretion, no changes were observed in the pattern of LH secretion which can account for the rescue of the corpus luteum.
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                Author and article information

                Contributors
                jdonato@icb.usp.br
                Journal
                Physiol Rep
                Physiol Rep
                10.1002/(ISSN)2051-817X
                PHY2
                physreports
                Physiological Reports
                John Wiley and Sons Inc. (Hoboken )
                2051-817X
                06 September 2022
                September 2022
                : 10
                : 17 ( doiID: 10.1002/phy2.v10.17 )
                : e15460
                Affiliations
                [ 1 ] Department of Physiology and Biophysics Instituto de Ciencias Biomedicas, Universidade de Sao Paulo Sao Paulo Brazil
                [ 2 ] Department of Anatomy Instituto de Ciencias Biomedicas, Universidade de Sao Paulo Sao Paulo Brazil
                Author notes
                [*] [* ] Correspondence

                Jose Donato Jr, Department of Physiology and Biophysics, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Av. Prof. Lineu Prestes, 1524, São Paulo, SP, 05508‐000, Brazil.

                Email: jdonato@ 123456icb.usp.br

                Author information
                https://orcid.org/0000-0002-0877-8453
                https://orcid.org/0000-0002-4166-7608
                Article
                PHY215460 PHY2-2022-08-0352
                10.14814/phy2.15460
                9446398
                36065891
                453fe2b9-c2a7-4766-a771-adfbf8ec6869
                © 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 15 August 2022
                : 22 August 2022
                Page count
                Figures: 9, Tables: 1, Pages: 18, Words: 9904
                Funding
                Funded by: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior , doi 10.13039/501100002322;
                Award ID: Finance Code 001
                Funded by: Fundação de Amparo à Pesquisa do Estado de São Paulo , doi 10.13039/501100001807;
                Award ID: 2016/20897‐3
                Award ID: 2017/22189‐9
                Award ID: 2019/21707‐1
                Award ID: 2020/01318‐8
                Award ID: 2020/10102‐9
                Award ID: 2021/03316‐5
                Categories
                Original Article
                Original Articles
                Custom metadata
                2.0
                September 2022
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.1.8 mode:remove_FC converted:06.09.2022

                follicle‐stimulating hormone,gnrh,hpg axis,kisspeptins,luteinizing hormone

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