Fig 2 - uploaded by Fatemeh Sabet Sarvestani
Content may be subject to copyright.
Effect of pregnancy on the relative expression of RFamide-related peptide-3 (RFRP-3) gene (mean ± SE) in the dorsomedial hypothalamic nucleus (DMH) rats (n=6 for each pregnancy day). a; Different letters indicate significant difference (p<0.01).
Source publication
Background: RFamide-related peptide-3 (RFRP-3) and kisspeptin are known to respectively inhibit and stimulate gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in rat. The aim of the present study was to evaluate the relative expression of RFRP-3 mRNA and KiSS-1 mRNA (the gene encodes kisspeptin) in the hypothalamus of pr...
Context in source publication
Context 1
... to RNX-Plus buffer (1 mL) in an RNase-free microtube, mixed thoroughly, and kept at room temperature for 5 minutes. Chlo- roform (0.2 mL) was added to the slurry and mixed gently. The mixture was centrifuged at 12,000 ×g (4 ̊C) for 20 minutes and the super - natant was transferred to another tube and pre- cipitated with an equal volume of isopropanol for 15 minutes. The RNA pellet was washed with 75% ethanol and quickly dried and re- suspended in 50 μL RNase-free water. The pu- rified total RNA was quantified by Nano-Drop ND 1000 spectrophotometer (Nano-Drop Tech- nologies, Wilmington, DE, USA). The DNase treatment was carried out using the DNase kit (Fermentas, St. Leon-Roth, Germany) accord- ing to the manufacturer’s instructions. The DNase-treated RNA (3 μg) was used for the first strand cDNA synthesis, using 100 pmol ol- igo-dT, 15 pmol dNTPs, 20 U RNase inhibitor, and 200 U M-Mulv reverse transcriptase (Fer- mentas, St. Leon-Roth, Germany) in a 20 μL fi- nal volume. Primers were designed using Allele ID 7 software (Premier Biosoft International, Palo Alto, USA) for the reference gene, KiSS-1 (NM_181692) and RFRP-3 (NM_023952). The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (M32599) was used as the ref- erence gene for data normalization (Table 1). Relative real-time PCR was performed in a 20 μL volume containing 1 μL cDNA, 1X Syber Green buffer and 4 pmol of each primer. The amplification reactions were carried out in a Line-Gene K thermal cycler (BIOER Technol - ogy Co., Ltd, Hangzhou, China) under the fol- lowing conditions: 2 minutes at 94 ̊C, 40 cycles of 94 ̊C for 10 seconds, 57 ̊C for 15 seconds, and 72 ̊C for 30 seconds. After 40 cycles, the specificity of the amplifications was tested by analyzing melting curves with the temperature ranging from 50 ̊C to 95 ̊C. All amplification reactions were repeated 3 times under identical conditions, including a negative control and 5 standard samples. To ensure that the PCR prod- ucts were generated from cDNA, but not the genomic DNA, proper control reactions were implemented in the absence of reverse tran- scriptase. For quantitative real-time PCR data, the relative expression of KiSS-1 was calculat- ed based on the threshold cycle (CT) method. The C T for each sample was calculated, using Line-gene K software (34). Accordingly, the fold expression of the target mRNAs over the reference values was calculated by the equa- tion 2- ΔΔCT (35), where ΔC T is determined by subtracting the corresponding GAPDH C T value (internal control) from the specific CT of the target (KiSS-1 or RFRP-3). The ΔΔC T was ob- tained by subtracting the ΔC T of each experi- mental sample from that of the control (ovariec- tomized rats). In Study 1, relationship between the cell char- acteristics of vaginal smear (proestrus, estrus, metestrus and diestrus) on days 4 and 5 after mating and positive and negative results of pregnancy were compared using the Chi-square test (SPSS for Windows, version 11.5, SPSS Inc, Chicago, Illinois). Percent of pregnant rats between three groups were analyzed using one- way analysis of variance (ANOVA) (SAS 9.1 SAS Institute Inc., Cary, NC). Tukey post hoc test was used for comparison of means within the groups. Values of p≤0.05 were considered significant. In Study 2, the data on relative expression of KiSS-1 and RFRP-3 genes were subjected to the test of normality and analyzed by one-way ANOVA, and mean separation was performed by Tukey’s test at p=0.01. Group means and their standard errors are reported in the text and figures (GraphPad Prism v 5.01, GraphPad software Inc., San Diego, CA, USA). In Study 1, there was a positive association be- tween pregnancy of rats and vaginal smear cell characteristics of metestrus (the same propor- tion among leukocytes, cornified and nucleated epithelial cells) or diestrous (a predominance of leukocytes) stages observed in days 4 and 5 after mating (p=0.001). If diestrous cell char- acteristics were observed in vaginal smear on day 4 after mating, and metestrous or diestrous cell characteristics were detected on day 5 after mating, 78.4% of rats would be pregnant. The cell characteristics of vaginal smear on days 4 and 5 were compared with cell characteristics of vaginal smear in different stages of estrous cycle in rat is shown in fig 1. Moreover, if me- testrous cell characteristics were observed on day 4 after mating and metestrous or diestrous cell characteristics were detected on day 5 af- ter mating, accuracy of pregnancy detection dropped to 60% which was not significantly different with the previous case (diestrous on day 4 and metestrous/diestrous on day 5). Only 16.7% of pregnant rats showed other cases of cellular characteristics of estrous cycle on days 4 and 5 after mating with chances to be pregnant was less than the two previous cases (p<0.05). In Study 2, the mean and standard error of rela- tive expression of RFRP-3 mRNA in DMH did not change during pregnancy (p>0.01, Fig 2). However, the relative expression of KiSS-1 mRNA in ARC was at its highest on day 7 of pregnancy and decreased until day 21 of pregnancy (p<0.01, Fig 3). The relative expression of KiSS-1 mRNA in ARC was at its peak in the first week of pregnancy and decreased 4-fold in the third week of preg- nancy in rat. In contrast to our results, Roa et al. (36) reported that KiSS-1 mRNA increased during the pregnancy in rat brains. In humans, kisspeptin levels increased by 940-fold in the first trimester in comparison with non-pregnant woman and further increased to some 7000-fold higher in the third tri- mester (37). Level of expression of KiSS-1 mRNA is higher in the first trimester placenta than in term placenta in humans (38), apparently contrasting with higher circulating kisspeptin levels reported during pregnancy (37). Considering the ability of systemically delivered KiSS-1 peptides to release LH (8, 39-42), this phenomenon seems to be at odds with the reported increase in serum kisspep- tin concentrations in human pregnancy (37). Al- though human and rat differ with regard to length of gestation and placental structure, the spatial and temporal expression of KiSS-1 mRNA are simi- lar. Kisspeptin and its receptors are detected in rat trophoblast (43). In specific, KiSS-1 mRNA is ex - pressed in the trophoblast giant cells of the rodent placenta (44), which are responsible for early inva- sion of spiral arteries and replacement of the endo- vasculature. These cells have the same functional phenotype as the human extravillous trophoblasts. As in humans, levels of KISS-1 and its receptor gradually decline during placental maturation and are not detectable at embryonic day 18.5 (44). The finding of the highest expression level of KISS-1 mRNA in trophoblast cells during the first trimes - ter in humans and at day 12.5 of pregnancy in ro- dents and decrease in day 15.5 to no detectable ex- pression on day 18.5 of pregnancy in rat coincides with the time of peak trophoblast invasion when regulation of this process is of critical importance (45, 46). It has been shown that the number of GnIH neurons have a positive correlation with plasma progesterone concentration (47) and that GnIH neurons are regulated by progesterone (48). Re- sponsiveness to RFRP-3 mRNA at pregnancy may derive from the combined exposure to high levels of progesterone and suppression of LH levels dur- ing pregnancy in rat (49). On the other hand, total cortisol and progesterone increased significantly from one trimester of pregnancy to the next in hu- mans (50). Increase of glucocorticoids caused an increase in RFRP that contributes to hypothalamic suppression of reproductive function in rat (51). Decrease of GnRH and LH secretion during rat pregnancy may be controlled by constant expres- sion of RFRP-3 mRNA and reduced expression of KiSS-1 mRNA. On the other hand, methods of early detection of pregnancy and exact determina- tion of pregnancy time in rats are widely applied on pregnant rats. Our presented method, in addi- tion to increasing the probability of rat pregnancy after the first mating event, can detect pregnancy with an accuracy of more than 60% on day 5. This research was financially supported by the Office of the Vice-Chancellor for Research, Shiraz University of Medical Sciences and the office of Student Scientific Affairs Manage- ment, Shiraz University. The authors declare no conflict of ...
Similar publications
RFamide-related peptide-3 (RFRP-3) may be involved in the inhibition of kisspeptin, but there is no direct evidence that RFRP-3 can directly act on kisspeptin neurons. The present study aimed to investigate the role and mechanism of RFRP-3 and kisspeptin in the hypothalamic-pituitary reproductive axis. In order to detect the expression and localiza...
Citations
... Before ovariectomy, vaginal smears were collected from all animals for 2 weeks to evaluate the regularity of their reproductive cycle using microscopic observation. The OVX was done during the estrus phase of the estrous cycle [48,49]. The rats were divided into several groups (n = 10), as shown in Table 1. ...
Objective:
Plant-derived estrogens (phytoestrogens) with structural similarity to primary female sex hormones could be suitable replacements for sex hormones. Therefore, the effects of the licorice root extract and Linum usitatissimum oil on biochemical and hormonal indices in the serum and uterine stereological changes in ovariectomized rats were evaluated.
Design:
In this study, 70 adult female rats were randomly divided into seven groups including 1) control group, 2) sham-operated group, 3) ovariectomized (OVX) group, 4) OVX rats that received 1 mg/kg estradiol for 8 weeks at the day of post-operation, 5) OVX rats which received 2.0 mg/kg body wt Linum usitatissimum oil for 8 weeks at the day of post-operation, 6) OVX rats which received 2.0 mg/kg body wt licorice extract for 8 weeks at the day of post-operation, and 7) OVX rats which received 2.0 mg/kg body wt Linum usitatissimum oil + 2.0 mg/kg body wt licorice extract for 8 weeks at the day of post-operation. After eight weeks, alkaline phosphatase activity, as well as calcium, estradiol, and progesterone concentrations were assessed and tissue samples of the uterus were serologically examined.
Results:
The results indicated that after 8 weeks of OVX the alkaline phosphatase activity (Mean = 637.7 IU/L) increased and the calcium (Mean = 7.09 mg/dl), estradiol (5.30 pmol/L), and progesterone (Mean = 3.53 nmol/L) reduced compared to other groups. Moreover, stereological changes in the uterus in ovariectomy groups were seen compared to the other groups. The treatment with Linum usitatissimum oil and licorice extract had a significant therapeutic effect on biochemical factors and stereological changes compared to the ovariectomized group.
Conclusion:
The results of this study showed that the combination of Linum usitatissimum oil with licorice extract showed the high potential of hormone replacement therapy in the reduction of OVX complications.
... Both Kp and GnIH are involved in pregnancy, as demonstrated in different species (Reynolds et al. 2009, Sabet Sarvestani et al. 2014, Martino et al. 2015. In particular, it has been reported that Kp levels increase by 940-fold in the first trimester, and further increase to around 7,000-fold higher in the third trimester, then fall to concentrations comparable with those prior to pregnancy, at 5 days post delivery, implicating a placental source of the peptide (Horikoshi et al. 2003). ...
Recently, two different molecules have been discovered to play an important role in reproduction: kisspeptin (Kp) and gonadotropin inhibiting hormone (GnIH). The aim of this study was to establish the trend of kisspeptin 10 (Kp‑10) and GnIH concentrations, during all phases of pregnancy in cattle, in order to understand their possible role in the physiology of pregnancy. To examine the correlation between these hormones and steroid hormones, cortisol and oestradiol 17β (E2) were also analyzed. Eighty pregnant cows were enrolled and the pregnancy was divided into 8 periods of 30 days each (from 30‑60 days to 240‑270 days). Blood samples were collected from all cows, once only for cow. Kp‑10, GnIH, cortisol and E2 were measured in sera. After an initial plateau, Kp‑10 concentrations increased at 90‑120 days and then decreased until 180‑210 days, undergoing a further increase until 240‑270 days. GnIH concentrations decreased until 90‑120 days, then increased until the end of gestation. These trends were opposing until 180‑210 days, whereat concentrations of both increased until the end of gestation. Cortisol concentrations were homogenous at all times, except at the final period, in which they were higher. E2 showed two peaks, at 90‑120 days and 240‑270 days. The trends in Kp‑10 and GnIH concentrations suggest that these two hormones might act to maintain the delicate endocrine equilibrium of pregnancy.
... Taking into account the observed differences appearing throughout the selected days of early pregnancy, an elevated abundance of Kiss and Kissr mRNA transcripts in the porcine anterior pituitary was only observed during the peri-implantation period, i.e. on days 15 to 16 of pregnancy. Interestingly, the increased Kiss mRNA transcript abundance was also demonstrated in the hypothalamic arcuate nucleus of rats during the first days of pregnancy [24]. Therefore, it cannot be excluded that conceptus-derived signals may be involved in the regulation of intra-pituitary mRNA transcript abundance, as was presented earlier in pigs during the peri-implantation period, i.e. in intra-pituitary steroid-response and estrogen synthesis, FSH secretion, immune response, PPAR signaling pathway, angiogenesis and potential for DNA methylation [42]. ...
... It is worth highlighting that, to date, knowledge regarding the RFRP-3/GPR147 system in the female pituitary gland during the estrous cycle is scarce [7,9,17], and its presence in the pituitary of pregnant females has not yet been demonstrated. The only available data documents an unchanged expression of Rfrp-3 mRNA transcript in the dorsomedial hypothalamic nucleus of pregnant rats [24]. The current study documented that the relative abundance of Rfrp-3 mRNA transcript in porcine anterior pituitary is increased during the peri-implantation period and luteolysis. ...
Kisspeptins (KISSs) and RFamide-related peptide-3 (RFRP-3) affect the synthesis and secretion of luteinizing hormone (LH) and modulate female reproductive processes. The presence of KISS and RFRP-3 in the porcine pituitary gland and their contribution to the regulation of follicle-stimulating hormone (FSH) synthesis and secretion is unknown. This study analyzed the presence of KISS and RFRP-3 in the pituitary of estrous-cyclic pigs on days 2 to 3, 10 to 11, 12 to 13, 15 to 16 and 19 to 20 and early pregnant pigs on days 10 to 11, 12 to 13 and 15 to 16, and evaluated the effect of KISS and RFRP-3 on β-Fsh mRNA expression and FSH secretion in vitro by pituitary cells collected on selected days of the estrous cycle. The cells were cultured in vitro and treated with KISS (10⁻⁶ M, 10⁻⁷ M) and RFRP-3 (10⁻⁶ M, 10⁻⁷ M) or gonadotropin-releasing hormone (GnRH; 100 ng/mL) alone and in combinations (4 h or 24 h). The relative abundance of Kiss and Rfrp-3 and their receptor mRNA transcripts, as well as the KISS and RFRP-3 proteins, were found in the pituitaries of estrous-cyclic and early pregnant pigs. KISS after 4 h increased the secretion of FSH in estrous cyclic pigs mostly during the early-luteal phase and luteolysis. RFRP-3 inhibited the synthesis and secretion of FSH in estrous-cyclic pigs on days 19 to 20 and the secretion of FSH on days 2 to 3 and 10 to 12 of the estrous cycle compared with GnRH-treated cells. KISS in co-treatment with GnRH after 24 h enhanced FSH release on days 2 to 3 and 15 to 16 of the estrous cycle. In conclusion, KISS and RFRP-3 systems are present in the pituitary of estrous-cyclic and pregnant pigs. In estrous-cyclic pigs, KISS and RFRP-3 may affect the synthesis and secretion of FSH by pituitary cells.
... Thereafter, their early pregnancy was confirmed using the vaginal smear method (31). In detail, the presence of vaginal plug in the female rats were examined in the next morning. ...
Background:
Animals can play an important role in preparing tissues for human through the development of xenotransplantation protocols. The most common problem with liver transplantation like any other organ transplantation is organ supply shortage.
Objective:
To evaluate the in utero xenotransplantation of mouse bone marrow-derived stromal/stem cells (BMSCs) to the liver of rat fetus to produce mouse liver tissue.
Materials and methods:
BMSCs were isolated and confirmed from enhanced green fluorescent protein (eGFP)-genetic labeled mice. Using a microinjection protocol, mice BMSCs were injected into the liver of rat fetuses in utero on day 14 of pregnancy. After birth, livers were collected and the presence of mice eGFP-positive cells in rat livers was evaluated through polymerase chain reaction.
Results:
The eGFP mRNA was detected in the liver of injected infant rats. BMSCs of adult mice were capable to remain functional probably as hepatocyte-like cells in liver of infant rats after in utero xenotransplantation.
Conclusion:
BMSCs have the potential for intrauterine xenotransplantation for the treatment of liver dysfunction before birth. This method can also be used for xenoproduction of liver tissue for transplantation.
... Moreover, prominent fibers have been seen in the ventral tegmental area, midbrain central gray, and dorsal motor nucleus of the vagus in the medulla in the zebra finch (Taeniopygia guttata) [25]. In the other investigations, GnIH neurons were exclusively expressed in the PVN, similar to the expression of GnIH neurons in mammalians, which were reported only in the PVN and dorsomedial area of goat [26] and rat [27,28]. The GnIH nerve fibers were distributed widely in the preoptic area, midbrain, and diencephalon of several avian species [21,25]. ...
... Aggregate amounts of nerve fibers in the median eminence and POA possibly may indicate the role of GnIH in the regulation of gonadotropin release. Similar findings were reported in mammals [27,28]. Approximately 80.5% of the GnRH-1 neurons were located in the POA [29], and 89.4% GnRH-2 neurons in the midbrain of European starlings [23]. ...
Gonadotropin-inhibitory hormone (GnIH), initially discovered in birds as a hypothalamic neuropeptide, inhibits the synthesis and release of gonadotropins via affecting the GnRH neurons and gonadotropes. Therefore, it could be a key neuropeptide in reproduction in birds. The aim of the present study was to investigate the pre-pubertal, pubertal and post-pubertal localization of GnIH and changes in hypothalamic GnIH expression in British United turkey hens. In pre-pubertal, pubertal and post-pubertal periods, the brains of turkey hens (n = 15) were removed after fixation. Sections (30 μm) were prepared from the entire hypothalamus and stained immunohistochemically against GnIH antibody. GnIH-immunoreactive neurons were observed in the paraventricular nucleus. These neurons were significantly more abundant in the pre-pubertal than pubertal and post-pubertal turkeys (P < 0.05). The results suggested that GnIH neurons have an important role in regulating the pubertal events in British United turkey-hens.
... In addition, the opposite expressions of kiss1 and GnIH mRNAs in the hypothalamus of rats during different stages of estrous cycle have been shown (76). This opposite expression of kiss1 and GnIH mRNAs in the hypothalamus has also been shown during malnutrition condition, pregnancy and lactation in female rats (77)(78)(79). RFRP-3/neuropeptide VF precursor (NPVF), cleaved from a preproprotein, is encoded by the NPVF (or RPFP) gene (80). In our previous study, induction of PCOS by continuous light reduced the mRNA expression of RFRP-3 neuropeptide in rats but did not have effect on kisspeptin expression (9,81). ...
Objective(s)
Polycystic ovary syndrome (PCOS) is an endocrinopathy in women, which, unlike its impact on fertility and health of women, there is no clear understanding about the causal mechanisms of this pathogenesis. The aim of this review paper is to investigate the pathophysiological pathways affecting the PCOS etiology, based on functions of gonadotropins– and steroid hormones–related genes.
Materials and Methods
Due to different hormonal and metabolic signs of this complex disorder, different hypotheses are mentioned about etiology of this syndrome. Because of the heterogeneity of the reasons given for this syndrome and the spread of the effective genes in its pathophysiology, most of genes affected by sex-related hormonal imbalances are examined for discriminative diagnosis. For this purpose, published articles and reviews dealing with genetic evaluation of PCOS in women in peer-reviewed journals in PubMed and Google Scholar databases were included in this review.
Results
In previous studies, it has been well demonstrated that PCOS in some individuals have a genetic origin. Pathophysiological functions of genes are primarily responsible for the synthesis of proteins that have role in PCOS before hyperandrogenism including GnRHR, FSHβ, FSHR, LHCGR, CYP19A1, HSD17B, AR and SHBG, and their effects in PCOS of human have been confirmed.
Conclusion
Hormonal imbalances are the first reason mentioned in PCOS etiology, and usually characterized with menstrual irregularities in PCOS women. Hyperandrogenism and gonadotropin secretion disorders are shown in PCOS condition, which are related to steroidogenesis pathways and hypothalamic–pituitary–ovarian axis disturbances, respectively.
... Given that it has been determined that serum LH tends to decrease to the lowest level at mid-pregnancy and recover at the end of pregnancy, it is not surprising that RFRP is associated with secretion of GnRH and LH (87). An emerging discovery is that the levels of GnRH and LH secretion were decreased in the hypothalamus of pregnant rats, promoting the hypothesis that GnRH and LH secretion may be controlled by transcription conditions of RFRP-3 and LH genes (87). ...
... Given that it has been determined that serum LH tends to decrease to the lowest level at mid-pregnancy and recover at the end of pregnancy, it is not surprising that RFRP is associated with secretion of GnRH and LH (87). An emerging discovery is that the levels of GnRH and LH secretion were decreased in the hypothalamus of pregnant rats, promoting the hypothesis that GnRH and LH secretion may be controlled by transcription conditions of RFRP-3 and LH genes (87). Similar to RFRP-3, kisspeptin is also a neuropeptide belonging to the large RFamide peptide subfamily. ...
Objective(s)
To benefit from reproduction and deal with challenges in the environmental conditions, animals must adapt internal physiology to maximize the reproduction rate. Maladaptive variations in the neurochemical systems and reproductive system can lead to manifestation of several significant mammalian reprocesses, including mammalian ovarian lifespan. RFamide-related peptide (RFRP, Rfrp), mammalian orthologues of gonadotropin-inhibitory hormone (GnIH), which is a regulator to prevent the gonadotropin-releasing hormone (GnRH) neural activity, is known to be related to reproductive traits. This review aimed to summarize recent five-year observations to outline historic insights and novel perspectives into the functions of RFRPs in coding the mammalian reproductive physiology, especially highlight recent advances in the impact on RFRPs in regulating mammalian ovary lifespan.
Materials and Methods
We reviewed the recent five-year important findings of RFRP system involved in mammalian ovary development. Data for this review were collected from Google Scholar and PubMed using the RFRP keyword combined with the keywords related to physiological or pathological reproductive functions.
Results
Recent discoveries are focused on three major fronts in research on RFRP role in female reproduction including reproductive functions, energy balance, and stress regulation. The roles of RFRPs in various development phases of mammal reproduction including prepuberty, puberty, estrous cycle, pregnancy, milking, menopause, and/or ovarian diseases have been shown.
Conclusion
Overall, these recent advances demonstrate that RFRPs serve as critical mediators in mammalian ovarian development.
... Our previous results with IHC method showed compacted populations of GnIH-ir neurons in the PVN of male partridges (Rezazadeh et al., 2017). GnIH and its mammalian orthologue, RF amide related peptide (RFRP) (Salehi et al., 2015), have role in different reproductive and physiological conditions including spermatogenesis (Karami Kheirabad et al., 2016), pregnancy (Sarvestani et al., 2014), lactation (Adavi et al., 2011;Asadi Yousefabad et al., 2013;Noroozi, Jafarzadeh Shirazi, Tamadon, Moghadam, & Niazi, 2015), estrus cycle (Jafarzadeh Shirazi, Namavar, & Tamadon, 2011a, 2011b, 2011cJafarzadeh Shirazi et al., 2013;Salehi et al., 2013), malnutrition condition (Jahanara et al., 2014), seasonal breeding Rezazadeh et al., 2017), and ovarian diseases (Shaaban et al., 2018). ...
Fast free of acrylamide clearing tissue (FACT) is a modified sodium dodecyl sulfate‐based clearing protocol for the chemical clearing of lipids that completely preserves fluorescent signals in the cleared tissues. The FACT protocol was optimized to image translucent immunostained brain and non‐nervous tissues. For this purpose adult male Chukar partridge (Alectoris chukar) was used as a model. After clearing the tissues, 1 or 2 mm‐thickness sections of tissues were immunolabeled. The paraventricular nucleus in the hypothalamus (2‐mm section) was cleared with FACT, and then was stained with gonadotropin‐inhibitory hormone (GnIH) antibody and Hoechst. Simultaneously, immunohistochemical (IHC) staining of cryosectioned brain (30 μm) was done by GnIH‐antibody. The FACT protocol and staining of cell nuclei of nine other tissues were done by a z‐stack motorized fluorescent microscope. GnIH‐immunoreactive neurons were found by FACT and IHC during the breeding season in male partridges. Deep imaging of the kidney, duodenum, jejunum, lung, pancreas, esophagus, skeletal muscle, trachea, and testis were also done. The FACT protocol can be used for the three‐dimensional imaging of various tissues and immunostained evaluation of protein markers.
Research highlight
• The FACT is a simple and cheap method for whole tissue clearing.
• The FACT‐cleared tissues can be imaged with simple fluorescent microscopes.
• For the first time, using the FACT, three‐dimensional imaging of various tissues was done.
... In addition, the opposite expression of kiss1 and GnIH mRNAs in the hypothalamus of rats during different stages of estrous cycle have been shown . This opposite expression of kiss1 and GnIH mRNAs in the hypothalamus has also been shown during malnutrition condition, pregnancy and lactation in female rats (Asadi Yousefabad et al., 2013;Jahanara et al., 2014;Sarvestani et al., 2014). RFRP-3/NPVF was cleaved from a preproprotein encoded by the NPVF (or RPFP) gene (Ayachi and Simonin, 2014). ...
... The distribution of RFRP-3 in the bovine hypothalamus, overall, matched well with descriptions of RFRP-3 distribution in the mouse (Ukena & Tsutsui, 2001), goat (Jafarzadeh Shirazi et al., 2014), sheep (Clarke et al., 2008), rat (Rizwan et al., 2009Yano et al., 2003), sparrow (Calisi, Rizzo & Bentley, 2008), hamster (Ubuka et al., 2012), o'possum (Harbid et al., 2013, and primate . Bovine RFRP-3 cells were located mainly in the DMN, but also distributed into the PVN and PrVN. ...
... In contrast, female non-human primates display lower neuropeptide VF precursor mRNA (the gene for RFRP in the primate) expression during the luteal phase than in the follicular phase of the menstrual cycle, and in ewes, the expression is reduced during the preovulatory period. . A recent report in the goat identified a greater number RFRP-ir cells in the DMN during the luteal stage compared to the follicular stage (Jafarzadeh Shirazi et al., 2014). The most profound and reproduced changes in RFRP-3 protein levels in the DMN have been in comparison of breeding and nonbreeding animals (Harbid et al., 2013;Smith et al., 2008a). ...
... Perhaps somewhat unique to previous reports, the current findings compared animals with equivalent estrogen levels and dissimilar progesterone levels. While not equivalent, in studies where RFRP-3 expression was measured in pregnant animals, in which progesterone levels were elevated, and nonpregnant animals the relative expression of RFRP-3 mRNA in DMN did not change (Sabet Sarvestani et al., 2014); suggesting dramatic changes in progesterone, alone, do not alter RFRP expression. ...
Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus (ARC) play a key role in gonadotropin-releasing hormone (GnRH) pulse generation and gonadal steroid feedback, with kisspeptin driving GnRH release and neurokinin B and dynorphin acting as pulse start and stop signals, respectively. A separate cell group, expressing RFamide-related peptide-3 (RFRP-3) has been shown to be a primary inhibitor of GnRH release. Very little is known regarding these cell groups in the bovine. In this study, we examined the relative immunoreactivity of kisspeptin, dynorphin, and RFRP-3 and their possible connectivity to GnRH neurons in the hypothalami of periestrus and diestrus bovine. While GnRH and RFRP-3 immunoreactivity were unchanged, kisspeptin and dynorphin immunoreactivity levels varied in relation to plasma progesterone concentrations and estrous status. Animals with higher plasma progesterone concentrations in diestrus had lower kisspeptin and increased dynorphin immunoreactivity in the ARC. The percentage of GnRH cells with kisspeptin or RFRP-3 fibers in close apposition did not differ between estrous stages. However, the proportions of GnRH cells with kisspeptin or RFRP-3 contacts (∼49.8% and ∼31.3%, respectively) suggest direct communication between kisspeptin and RFRP-3 cells to GnRH cells in the bovine. The data produced in this work support roles for kisspeptin and dynorphin, within the KNDy neural network, in controlling GnRH release over the ovarian cycle and conveying progesterone-negative feedback onto GnRH neurons in the bovine.
