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Background Matrix-producing breast carcinoma (MPBC) is a very rare and usually aggressive triple-negative breast cancer. We successfully established a patient-derived orthotopic xenograft (PDOX) model from a patient with MPBC and used it to study tumor sensitivity to various agents. Case report A 40-year-old woman was diagnosed with MPBC with a triple-negative phenotype. Due to axillary lymph-node metastases found during radical mastectomy, the patient was subsequently treated with epirubicin, cyclophosphamide and paclitaxel. In addition, radiotherapy was directed to the chest wall and subclavicular fossa. A portion of the cancer tissue from the mastectomy was used to establish a PDOX nude-mouse model. The PDOX model was resistant to paclitaxel, bevacizumab, vinorelbine, cisplatinum and olaparib, and sensitive to eribulin. Metastases in mediastinal lymph nodes and the right ovary were observed in the patient 14 months after mastectomy. Thoracoscopic mediastinal lymph-node biopsy and laparoscopic oophorectomy were performed, and both confirmed breast-cancer metastasis. The patient was then treated with paclitaxel and bevacizumab but no response was observed, which correlated with the inability of these drugs to arrest tumor growth in the PDOX models of the patient's tumor. The patient was then given eribulin based on the PDOX-model result, but treatment had to be stopped because of rapid progression of metastasis into the cervical lymph nodes and thyroid gland. The patient was subsequently treated with atezolizumab and nab-paclitaxel. Unfortunately, the patient died of her cancer 8 months after recurrence. Conclusion The present study demonstrates that the PDOX model of a patient's triple-negative MPBC accurately predicted that paclitaxel and bevacizumab would not arrest the patient's tumor growth. Eribulin may have been effective if administered at an earlier stage of the patient's cancer course. Drug-screening results from PDOX models should be used as early as possible in order to improve patient outcome.

Background/aim Matrix-producing breast carcinoma (MPBC) is a rare and usually aggressive triple-negative breast cancer (TNBC). In the present report, we determined the drug sensitivity for a triple-negative MPBC using a patient-derived orthotopic xenograft (PDOX) model. Materials and methods The PDOX model was established in the left 2nd mammary by surgical orthotopic implantation (SOI). MPBC PDOX models were randomized into 4 groups (6 mice per group) when the tumor volume became 80 mm3: G1, control group; G2, cisplatinum group [intraperitoneal (i.p.) injection, weekly, for 2 weeks]; G3, paclitaxel group (i.p., weekly, for 2 weeks); G4, eribulin group [intravenous (i.v.) injection, weekly, for 2 weeks]. All mice were sacrificed on day 15. Tumor volume and body weight were measured one time per week. Results The MPBC PDOX model was resistant to cisplatinum (p=0.800). Paclitaxel suppressed tumor growth compared to the control group (p=0.009). However, only eribulin regressed the tumor (p=0.001). Conclusion Eribulin has clinical potential for triple-negative MPBC patients.

Gonadotropin-releasing hormone (GnRH) stimulation of annexin A1 (ANXA1) and A5 (ANXA5) mRNA expression was analyzed in LβT2 gonadotrope cells. Quantitative polymerase chain reaction results showed that a GnRH analog (GnRHa) stimulated the expression of both ANXA1 and A5 mRNA with a peak at 12 h of incubation; however, ANXA1 mRNA was extremely stimulated (60 folds). Immunocytochemical analysis confirmed these findings. A GnRH antagonist inhibited the effect of GnRHa. ANXA1 and A5 mRNA levels were significantly increased by protein kinase C (PKC) activator (12-O-Tetradecanoylphorbol-13-acetate; TPA), but not by dibutyryl cAMP. GnRHa-stimulated induction of ANXA1 and A5 mRNA was inhibited by PKC (GF109203) and MEK inhibitors (PD98059). TPA increased ANXA1 and A5 mRNA expression in a dose-dependent manner (1 nM to 10 μM), while the extent of the increase was much greater in ANXA1. After stimulation with 10 nM or 1 μM TPA, ANXA1 and A5 mRNA levels were increased at 6 h. ANXA1 mRNA levels were higher in the 1 μM TPA than in the 10 nM TPA treatment, whereas 1 μM TPA did not show further stimulation of ANXA5 mRNA compared to 10 nM TPA. These results clearly show that ANXA1 mRNA expression is stimulated by GnRH through PKC like ANXA5, and the response of ANXA1 is much larger than that of ANXA5. A close relationship between these annexins and a significant role for ANXA1 in GnRH action at gonadotropes is suggested.

Cervical cancer is a recalcitrant disease. To help overcome this problem, we previously established a patient-derived orthotopic xenograft (PDOX) model of cervical cancer. In the previous study, we found the tumor to be resistant to nab-paclitaxal (nab-PTX). We also previously developed the tumor-targeting bacteria Salmonella typhimurium A1-R (S. typhimurium A1-R). The aim of the present study was to investigate the efficacy of S. typhimurium A1-R to overcome nab-PTX resistance in the cervical cancer PDOX model. Cervical-cancer tumor fragments were implanted orthotopically into the neck of the uterus of nude mice. The cervical-cancer PDOX models were randomized into the following four groups after the tumor volume reached 60 mm3: G1: untreated group; G2: nab-PTX (i.v., 10 mg/kg, biweekly, 3 weeks); G3: Salmonella typhimurium A1-R (i.v., 5 × 107 CFU/body, weekly, 3 weeks); G4: nab-PTX combined with Salmonella typhimurium A1-R (nab-PTX, 10 mg/kg, i.v., biweekly, 3 weeks; S. typhimurium A1-R, 5 × 107 CFU/body, i.v., weekly, 3 weeks). Each group comprised eight mice. All mice were sacrificed on day 22. Tumor volume was measured on day 0 and day 22. Body weight was measured twice a week. Nab-PTX and Salmonella typhimurium A1-R did not show significant efficacy as monotherapy compared to the control group (P = 0.564 and P = 0.120, respectively). In contrast, nab-PTX combined with Salmonella typhimurium A1-R significantly suppressed tumor growth compared to the untreated control group and nab-PTX group (P

Background/aim Matrix-producing breast carcinoma (MPBC) is a rare and usually aggressive triple-negative breast cancer (TNBC). In this study, we determined drug sensitivity for a triple-negative MPBC, without BRCA mutations, in a patient-derived orthotopic xenograft (PDOX) model. Materials and methods The MPBC PDOX model was established in the left 2nd mammary gland of nude mouse by implantation of the patient tumor using surgical orthotopic implantation (SOI). We randomized MPBC PDOX mice into 5 groups (n=5 mice/per treatment group) when the tumor volume reached 80 mm3: G1, control-no treatment; G2, bevacizumab [intra-peritoneal (i.p.), weekly, for 2 weeks]; G3, vinorelbine (i.p., weekly, for 2 weeks); G4, olaparib (oral., daily, for 2 weeks); G5, eribulin [intravenous (i.v.), weekly, for 2 weeks]. The mice in each treatment group were sacrificed on day 15. Tumor volume and body weight were measured once/week. Results The MPBC PDOX model was resistant to olaparib (p=0.22). The MPBC PDOX model treated with bevacizumab and vinorelbine showed significantly suppressed tumor growth compared to the untreated group (p=0.005 and 0.002, respectively). However, only eribulin regressed the tumor (p=0.0001). Eribulin was more effective than olaparib (p=0.0001), bevacizumab (p=0.0025) and vinorelbine (p=0.0061). Conclusion Eribulin has clinical potential as treatment for triple-negative MPBC patients that are resistant to a PARP inhibitor such as olaparib.

Estrogen action is mediated through several types of receptors (ERs), such as ERα, ERβ and putative membrane ERs. Oxytocin receptor (OTR) and ER expression levels in the rat uterus are regulated by estrogen; however, which types of ERs are involved has not been elucidated. This study examined OTR, ERα and ERβ levels in ovariectomized rats treated with 17β-estradiol (E2), an ERα agonist (PPT), an ERβ agonist (DPN) or estren (Es). E2 and PPT increased OTR mRNA levels and decreased ERα and ERβ mRNA levels 3 and 6 h posttreatment. DPN decreased ERα and ERβ mRNA levels at 3 and 6 h, while OTR mRNA levels increased at 3 h and decreased at 6 h. OTR mRNA levels increased 3 h after the Es treatment and then declined until 6 h. ERα and ERβ mRNA levels decreased by 3 h and remained low until 6 h posttreatment with Es. The ER antagonist ICI182,780 (ICI) suppressed the increases in OTR mRNA levels induced 3 h after the Es treatment. However, ICI and tamoxifen (Tam) had no significant effect on ERα and ERβ mRNA levels in the Es-treated or vehicle-treated group. In intact rats, proestrus-associated increases in OTR mRNA levels were antagonized by both ICI and Tam. However, decreases in ERα and ERβ mRNA levels were not antagonized by Tam and ICI, respectively. Therefore, uterine OTR gene expression is upregulated by estrogen through the classical nuclear (or non-nuclear) ERs, ERα and ERβ, while the levels of these ERs are downregulated by estrogen through multiple pathways including Es-sensitive nonclassical ERs.

Background: Cervical cancer is a world-wide problem that requires transformative therapeutic strategies. We have previously developed patient-derived orthotopic xenograft (PDOX) nude-mouse models of this disease. In the present report, we demonstrate that the standard drug, cisplatinum (CDDP), is highly-effective while the new, highly-touted agent, nab-paclitaxel (NAB-PTX) is ineffective. Materials and Methods: Cervical PDOX tumors were grown on the cervix of nude mice for 4 weeks after surgical orthotopic implantation (SOI). Tumors were treated with CDDP or NAB-PTX. Results: H&E staining demonstrated that the PDOX tumor recapitulated the original patient tumor. CDDP was highly-effective. One tumor that was treated with CDDP completely regressed. CDDP-treated tumors were smaller (tumor volume ratio: 0.42±0.36) than the control group (tumor volume ratio: 3.47±1.66) (p

We describe here patient-derived orthotopic xenograft (PDOX) models of cervical cancer in nude mice. The PDOX models metastasized in a similar pattern as the patient, including lymph nodes, liver, lungs and peritoneum. Most importantly, the drug, entinostat, was active against the metastasis and not the primary tumor or a subcutaneous model, which did not metastasize. In another cervical cancer PDOX, the generic drug, cisplatinum, was active in comparison with an expensive patented drug, NAB-paclitaxel, which was not. The models described in this chapter should be of use to individualize and make more precise and effective treatment for patients with metastatic cervical cancer.

Both neuropeptide Y (NPY) and ghrelin can enhance the feeding behavior. The purpose of this study was to determine whether NPY and ghrelin are involved in hyperphagia and obesity induced by lesions of the hypothalamic paraventricular nucleus (PVN).Sham-operated control rats and rats subjected to bilateral electrolytic lesions of the PVN were administered NPY (5 μg/rat) by intracerebroventricular infusion or ghrelin (20 μg/kg) by intraperitoneal (i.p.) injection. Control rats were administered the appropriate vehicle by the same route as the drug. We measured the cumulative food intake (FI) for 2 h after infusion of NPY and for 4 h after ghrelin injection.NPY significantly increased the cumulative FI in sham-operated rats. In PVN-lesioned rats, however, the cumulative FI at each time point (15 min, 30 min, 1 h, and 2 h) after NPY infusion was not significantly different from vehicle infusion, showing that NPY lost its orexigenic effect in PVN-lesioned rats. Following ghrelin injection, the cumulative FI was greater in PVN-lesioned rats than sham-operated rats, indicating that PVN lesions enhanced the orexigenic effects of ghrelin.These results suggest that the hyperphagia and obesity induced by PVN lesions may be related to an increased orexigenic action of ghrelin, but not NPY.

Leptin has been thought to work as a mediator for body weight control by inhibiting food intake. Leptin, however, cannot prevent obesity induced by a high-fat diet (HFD) probably because of leptin resistance. We investigated daily feeding and weight gain when ordinary chow (OC) was changed to a HFD in male rats. Food intake, by weight, significantly increased the next day, but gradually decreased until at 20 days the HFD intake contained the same calories as consumed by the OC-fed control rats. The reduction in food intake occurred only during the night without change of preference for the HFD, even after leptin resistance had developed. Nonetheless, the HFD-fed rats gained more weight than the controls. From the present experiment, it is concluded that leptin resistance does not induce hyperphagia, and suggested that body weight is not regulated to be constant.

Estrogen inhibits food intake in cycling females in a variety of species. To determine how the development of the anorexic system by estrogen is regulated, rat pups at four developmental stages, postnatal day 11 (P11)-13, P20-22, P25-27 and P29-31, and adult ovariectomized (OVX) rats received a daily subcutaneous injection of 20 µg/kg of estradiol benzoate (EB) or vehicle for three days. Food intake, body weight gain and immunohistochemical c-Fos expression in the brain were measured after each injection. EB treatment decreased both food intake and body weight gain from P27 onwards and significantly increased c-Fos expression in the parvocellular division of the paraventricular nucleus of the hypothalamus (pPVN), which is coincident with its anorexic effect in developing rats. The pattern of EB-induced c-Fos activation in other feeding-related nuclei did not coincide with its anorexic effect in developing pups. However, in adult OVX rats, EB treatment increased c-Fos expression in the nucleus tractus solitarius (NTS), the central nucleus of the amygdala (CeA), and, to a lesser degree, the ventromedial nucleus of the hypothalamus (VMH). These results suggested that the pPVN is an essential site in the brain for controlling the anorexic effect of estrogen and that the feeding system of rat begins to respond to estrogen before the onset of puberty (P25-28).

Orexins are endogenous neuropeptides that potently facilitate appetite and food consumption. In the present study, we examined orexin immunoreactivity and prepro-orexin mRNA expression in the lateral hypothalamus by immunohistochemistry and competitive reverse transcription-polymerase chain reaction (RT-PCR) methods in different models of hyperphagia in rats. Hyperphagia was induced by lesions of either the ventromedial hypothalamus (VMHL) or the paraventricular nucleus (PVNL), and we also compared lactating rats to nonlactating controls. Both VMHL and PVNL increased food intake and body weight compared to shams. On day 7 post lesion, serum leptin and insulin concentrations exhibited 3.2- and 2.8-fold increases in VMHL rats, and nonsignificant 1.8- and 1.8-fold increases in PVNL rats; there were significant decreases (48% and 33%) in lactating rats on day 12 postpartum compared to controls, respectively. Serum glucose concentrations were not significantly changed compared to controls in these rats. Quantification by image analysis suggests that VMHL significantly decreased the number and mean staining intensity of orexin-A immunoreactive neurones compared to those in the sham-lesioned group; while PVNL did not change orexin-A immunoreactivity. Competitive RT-PCR analysis showed that VMHL significantly decreased the prepro-orexin mRNA expression compared to those in the sham-lesioned group, and PVNL did not change it. Lactating rats on days 11-12 of lactation had significantly greater number and mean staining intensity of orexin-A immunoreactive neurones, prepro-orexin mRNA expression food intake and body weight than nonlactating postpartum rats. Thus, changes in orexin-A immunoreactivity and prepro-orexin mRNA expression were not consistent between the hyperphagia models. These results suggest that the hyperphagia from VMHL or PVNL and lactating rats differ in their involvement of orexin-A, and the change in circulating leptin and insulin concentrations may be involved in the change of orexin-A immunoreactivity in these rats.

Oxytocin receptor (OTR) mRNA levels in the uterus dramatically increase in the near term human and rat. Estrogen is believed to be a potent stimulator of OTR mRNA expression. However, estrogen does not stimulate rat OTR mRNA expression on day 18 of pregnancy or in progesterone-treated rats. Thus, the regulation of uterine responsiveness to estrogen in the near term rat appears to be an important mediator of estrogen action. To determine the effect of altering uterine responsiveness to estrogen on OTR induction, uterine ERalpha and ER beta mRNA levels were examined by competitive RT-PCR in pregnant and parturient rats, progesterone-treated ovariectomized (OVX) virgin rats and OVX pregnant rats. In pregnant and parturient rats, OTR mRNA levels were highest at 2200-2230 h on day 21 of pregnancy (P21pm) and during labor when compared with other groups. ERalpha mRNA levels significantly increased during labor compared with days 15-21 of pregnancy. Compared with control animals, ERalpha mRNA levels decreased significantly in OVX virgin rats implanted with tubes containing progesterone for one week; 24 h following the removal of the progesterone tubes, ERalpha mRNA levels were found to be similar to control levels. Estrogen treatment following OVX on day 18 of pregnancy caused increased OTR mRNA levels, whereas ovariectomy alone increased ERbeta mRNA but not ERalpha mRNA. Results from the present study suggest that ERalpha and ERbeta mRNA expressions are differentially regulated in the rat uterus. Moreover, during spontaneous labor our findings appear to suggest that ERalpha plays a more prominent role than ERbeta in mediating estrogen action in the induction of uterine OTR mRNA before labor.

We have previously shown the involvement of the ventromedial nucleus of the hypothalamus (VMH) in inducing running behavior. Stimulation of kainate (KA)-type glutamate receptors in the unilateral VMH of the rat exclusively elicited stereotyped running behavior. However, the neural pathways or functional connections of the VMH neurons involved in the running activity are yet to be elucidated further. In this study we examined whether the subthalamic locomotor region (SLR) is involved in the expression of the running activity originating in the VMH. The multiunit activity (MUA) in the ipsilateral SLR was significantly increased by KA injection into the VMH of urethane-anesthetized animals. Concomitant injection of 6,7-dinitroquioxalline-2,3-dione (DNQX, a KA-type glutamate receptor antagonist) with KA blocked this change in the MUA. Unilateral pre-injection of either kynurenate (non-selective glutamate receptor antagonist), D-2-amino-5-phosphonovalerate (AP5, an NMDA-type glutamate receptor antagonist) or DNQX into the SLR blocked the expression of the running activity induced by KA injection into the ipsilateral VMH. Results from the present study suggest that communication between KA-sensitive efferents from the VMH to glutamatergic pathways acting via NMDA and non-NMDA receptors in the SLR may underlie expression of running behavior originating in the VMH.

The present experiments were undertaken to examine whether leptin affects the electrical activity of neurones in the supraoptic nucleus (SON) by using brain slice preparation of male Wistar and obese Zucker rats. Bath application of leptin (10(-8) - 10(-12) M) induced mainly inhibitory response in SON neurones of Wistar rats, although a minority showed excitation. These effects were observed in both continuously and phasically active cells. The inhibitory effect of leptin still persisted in low Ca(2+), high Mg(2+) medium. Bath application of tolbutamide, which is known to inhibit ATP-sensitive potassium channel activity, did not reverse the inhibitory effect of leptin on SON neurones. The effect of bath application of leptin was also tested in SON neurones of obese Zucker rats. Although leptin still affected the electrical activity of some SON neurones of Zucker rats, the proportion of unaffected neurones was significantly higher than in Wistar rats. The results suggest that leptin may inhibit the secretion of both oxytocin and vasopressin by inhibiting the electrical activity of neurones in the SON via direct action. This inhibitory effect of leptin may be exerted through mechanisms other than activation of ATP-sensitive potassium channels.

Wnt/β-catenin signalling regulates numerous developmental and homeostatic processes. Ctnnb1 (also known as β-catenin) is the only protein that transmits signals from various Wnt ligands to downstream genes. In this study, we report that our newly established mouse strain, which harbours a Cys429 to Ser missense mutation in the β-catenin gene, exhibited specific organ defects in contrast to mice with broadly functioning Wnt/β-catenin signalling. Both homozygous mutant males and females produced normal gametes but were infertile because of abnormal seminal vesicle and vaginal morphogenesis. An ins-TOPGAL transgenic reporter spatiotemporally sustained Wnt/β-catenin signalling during the corresponding organogenesis. Therefore, β-cateninC429S should provide new insights into β-catenin as a universal component of Wnt/β-catenin signal transduction.

This study examined the effect of suckling on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical marker for nitric oxide synthase, NOS) reactivity and neuronal NOS mRNA expression in the paraventricular (PVN) and supraoptic (SON) nuclei of lactating rats. Freely nursing (non-separated) dams and those separated from pups for 12 h and then reunited for 0, 15, 30, 60, 90, 120 and 180 min were used for the study. Dams separated from pups and sacrificed at time zero (without reunion) showed a significant decrease in NADPH-d staining and NADPH-d positive cells as well as in the NOS mRNA expression in the PVN and SON compared to that observed in non-separated dams. Reunion with pups and restoration of suckling significantly increased NADPH-d reactivity after 15, 30, 60 min, but not after 90, 120 and 180 min compared to non-reunited pups-deprived dams. A pattern of NADPH-d reactivity and neuronal NOS mRNA expression indistinguishable from that observed during free lactation was reinstated shortly (15 min) after the restoration of suckling stimulus, suggesting that the NADPH-d reactivity in lactation depends on the presence of the suckling stimulus. These results show that suckling stimulus may play a modulatory role in the regulation of NOS reactivity in the magnocellular neurones of the hypothalamic PVN and SON during lactation.

Spontaneously hypercholesterolemic (SHC) rats are known to exhibit accelerated bone resorption. We compared endochondral bone formation induced by implantation of demineralized bone matrix (DBM) to 4-week-old SHC rats with that of age-matched Sprague-Dawley (SD) rats. When DBM prepared from adult SD rats was implanted, the cartilageous area enlarged, and C-propeptide of type II procollagen content on day 7 was higher in SHC rats. Alkaline phosphatase activity and calcium content on day 12 and tartrate-resistant acid phosphatase activity on day 19 were higher in SHC rats. These results suggest active chondrogenesis, with a subsequent increase in osteogenesis, and stimulated osteoclastic bone resorption in SHC rats. When DBM from 10-week-old SHC rats was implanted into SD or SHC rats, the levels of bone forming parameters on day 12 were reduced to one-third, suggesting inhibiting factor(s) for bone induction in bone matrix of SHC rats. In contrast, when DBM from 6-month-old SHC rats was implanted, although bone forming parameters in SD rats were comparable to the case of implantation of DBM from SD rats, the accelerated bone formation detected in SHC rats was blocked, indicating resistance to systemic bone inducing factor(s) of SHC rats in aged bone matrix. These results suggest that age-related decrease in responses to some systemic bone inducing factor may lead to the bone loss with advancing age.

We have studied immunolocalization of activin receptors in the central nervous system using polyclonal antibodies (IgG) to type I (50-55 kDa, ActRI), type II (70-75 kDa, ActRII) or a subtype of type II known as type IIB (ActRIIB) receptors of activin. A total of 7 antisera to rat activin receptors was generated, i.e. 3 kinds of antisera to the extracellular domain (ActRI(81-89), ActRII(91-100), or ActRIIB(90-99)) and 4 antisera to the kinase domain (ActRI(323-333), ActRII(307-319), ActRII(407-420) or ActRIIB(306-319)). The region of aa 407-420 of ActRII is identical with that of ActRIIB. At first, we characterized these antibodies by Western blot analysis using ovarian proteins fractionated by preparative SDS-PAGE. All antibodies to ActRII and ActRIIB specifically reacted with 75 kDa-proteins which could also bind to activin-A. Anti-ActRII(91-100) antibody also reacted with 62 kDa-proteins which were capable of binding with activin-A. Although no positive reactions to anti-ActRI(81-89) antibody were seen in ovarian proteins, a positive reaction was detected at 52 kDa only when the proteins were deglycosylated. By use of these antibodies, immunolocalization of activin receptors was examined in the rat brain. The patterns of expression of activin type I and type II receptors were different. Positive reactions to anti-ActRII(91-100) antibody were detected in neurons of the cerebral cortex, hippocampus, medial amygdala and thalamus. In the hypothalamus, some neurons of the supraoptic nucleus were weakly stained, and widely scattered neurons of the lateral hypothalamic area were moderately stained. On the contrary, the most intense reactions to anti-ActRI(81-89) antibody were detected in neurons of the lateral hypothalamic area. In addition, many neurons of the cerebral cortex were also stained, but neurons of the hippocampus and the amygdala were not stained. These results suggest that activin may have physiological roles not only for hypothalamic neuroendocrinological and feeding-related systems as suggested previously but may also have functions in cortical and limbic pathways as a neuromodulator or for maintenance of neurons.

This study examined the effect of nitric oxide (NO) on milk transfer in rats. Pups nursed by mothers that received chronic systemic injections of sodium nitroprusside (SNP) weighed significantly less than pups of mothers treated with either saline or N omega-nitro-L-arginine (NNLA). Intracerebroventricular injection of SNP or L-arginine (L-arg) but not NNLA or saline, caused a significant reduction of milk transfer from mother to pups after a 12 h separation period. Systemic oxytocin (OT) injection reversed the effect of central injection of SNP. Furthermore, SNP and L-arg inhibited, whereas NNLA permitted the characteristic milk ejection burst of OT neurones without changing myoepithelial tissue response to systemic OT. These observations suggest that NO may be involved in the regulation of milk ejection bursts and milk transfer.

The involvement of activin and follistatin, an activin-binding protein, in endochondral bone development was examined by sc implantation of demineralized bone matrix in rats. Immunoreactive follistatin was localized in proliferating chondrocytes and round osteoblasts, whereas it was not detected in hypertrophic chondrocytes and osteoblasts surrounding bone marrow. Western blot analysis also revealed that immunoreactive follistatin was higher during the initial stages of chondrogenesis (day 5) and osteogenesis (days 11 and 14) and lower during the conversion from cartilage to bone (day 9). These results suggest that follistatin is produced by proliferating cells, and the expression decreases with differentiation of the cells. Implants injected with follistatin on days 9 and 10 contained lower calcium levels on day 14 than those injected with rat albumin. Furthermore, the follistatin-injected implants were still mainly composed of cartilage, suggesting that the disappearance of follistatin is necessary for the conversion of cartilage to bone. In contrast, immunoreactive activin beta A (55-60 kDa) was continuously detected in implants on days 7-14. The content of C propeptide of type II procollagen was increased and cartilageous area was enlarged on day 7 by activin A injections on days 5 and 6, suggesting a chondrogenic effect of activin in the initial stage of cartilage formation. These results indicate that proliferating chondrocytes and round osteoblasts produce follistatin, and that the activity of activin is regulated by changes in the expression of follistatin at the stages of chondrogenesis and transition from cartilage to bone.

Reproductive functions are regulated by the sophisticated coordination between the neuronal and endocrine systems and are sustained by a proper nutritional environment. Female reproductive function is vulnerable to effects from dietary restrictions, suggesting a transient adaptation that prioritizes individual survival over reproduction until a possible future opportunity for satiation. This adaptation could also partially explain the existence of amenorrhea in women with anorexia nervosa. Because amino acid nutritional conditions other than caloric restriction uniquely alters amino acid metabolism and affect the hormonal levels of organisms, we hypothesized that the supply of essential amino acids in the diet plays a pivotal role in the maintenance of the female reproductive system. To test this hypothesis, we examined ovulatory cyclicity in female rats under diets that were deficient in threonine, lysine, tryptophan, methionine or valine. Ovulatory cyclicity was monitored by daily cytological evaluations of vaginal smears. After continuous feeding of the deficient diet, a persistent diestrus or anovulatory state was induced most quickly by the valine-deficient diet and most slowly by the lysine-deficient diet. A decline in the systemic insulin-like growth factor 1 level was associated with a dietary amino acid deficiency. Furthermore, a paired group of rats that were fed an isocaloric diet with balanced amino acids maintained normal estrous cyclicity. These disturbances of the estrous cycle by amino acid deficiency were quickly reversed by the consumption of a normal diet. The continuous anovulatory state in this study is not attributable to a decrease in caloric intake but to an imbalance in the dietary amino acid composition. With a shortage of well-balanced amino acid sources, reproduction becomes risky for both the mother and the fetus. It could be viewed as an adaptation to the diet, diverting resources away from reproduction and reallocating them to survival until well-balanced amino acid sources are found.

When a L-lysine (Lys)-deficient diet is given to rats, Lys in plasma and brain declines and rats will then select a Lys solution from among other L-amino acids (AAs). The recording of single-unit activity in the lateral hypothalamic area of these rats suggested that neural plasticity occurred, specifically responding to the deficient nutrient, Lys, centrally and during ingestion of AA. Possible neurotrophic factors in serum from rats with or without deficiency of either protein or Lys was assayed by Hydra japonica. An increase in serum inhibin and activin A was observed in rats fed a Lys-sufficient and nonprotein diet, respectively. However, serum activin A-like activity was severely suppressed under Lys deficiency. Additionally, the immunohistochemical distribution of activin A in the brain was found in the nucleus tractus solitarius, the area postrema, and the arcuate nucleus. These facts indicate that ingestion of Lys-deficient or nonprotein diet caused a change in serum levels of activin A as a possible neurotrophic factor. This release may elicit plasticity in the sensitivity of neurons to deficient AA in the nuclei that could selectively drive ingestive behavior for its particular AA (e.g., Lys) to maintain AA homeostasis.

Incubations of rat anterior pituitary cells with transforming growth factor (TGF)-beta 1 for 48 hr suppressed the secretion of basal prolactin (PRL) in a dose-dependent manner (ED50, 100 pg/ml). Activin, a gonadal hormone processing cysteine distribution similar to TGF beta, also suppressed basal PRL secretion, but it was less effective (ED50, 4 mg/ml). Treatment with TGF beta 1 significantly suppressed basal PRL secretion from the pituitary after 24 hr and up to 72 hr of incubation. TGF beta 1 also inhibited thyrotropin-releasing hormone-mediated PRL secretion and activin inhibited thyrotropin-releasing hormone-mediated PRL secretion slightly, but significantly. In addition, we also measured the secretion of growth hormone by cultured pituitary cells treated with TGF beta 1 or activin for 24 to 72 hr. TGF beta 1 and activin showed an opposite effect on growth hormone secretion; TGF beta stimulated and activin inhibited basal secretion of growth hormone. These results suggest that TGF beta 1 is a potent inhibitor of basal secretion of PRL by the pituitary, and both TGF beta 1 and activin play a multifunctional role in basal secretion of pituitary hormones.

The relationship between prolactin (PRL) secretion and anovulation in lactating rats was studied. Normal lactating rats and lactating rats treated with antiserum against luteinizing hormone-releasing hormone at the time of postpartal ovulation were used. Normal lactating rats were treated with either a dopamine agonist (CB-154, 150 micrograms/rat) on Day 10 or 13, or pups removal on Day 7 or 10, and thereafter luteolysis and inhibition on PRL secretion were assessed. With the CB-154 treatment, the incidence of luteolysis increased as the lactational period advanced (42% vs 72%), whereas it decreased (73% vs 14%) with the pups removal. Thus, dopamine effectively inhibited PRL secretion during the later lactational stage, but could not do so during the earlier stage when there were mechanisms other than dopamine stimulating PRL secretion. Following luteal regression induced by CB-154, ovulation did not occur if the rats were treated with CB-154 on Day 10, whereas 50% of the rats ovulated within 4 days if treated on Day 13. Furthermore, in the lactating rats treated with anti-luteinizing hormone-releasing hormone serum during late pregnancy, ovulation was not observed until Day 10 of lactation. Since the serum progesterone levels were low in these rats due to the absence of ovulation and lactational corpora lutea, the blockade of ovulation was not due to elevated circulating progesterone during the early lactational period. The mechanism of ovulation blockade during lactation thus seems to shift from being progesterone independent to progesterone dependent at a similar period when the neuroendocrine control of PRL secretion shifts from dopamine independent to dependent.

Interleukin-1 beta (IL-1 beta) at doses of 0.15 and 1.5 nM significantly inhibited FSH secretion and stimulated LH secretion by cultured rat pituitary cells after 24-72 hr incubation whereas 15 pM of IL-1 beta was not effective. Treatment with IL-1 beta for 12-48 hr did not affect intracellular content of FSH. However, treatment with 0.15 and 1.5 nM of IL-1 beta for 72 hr significantly suppressed intracellular content of FSH whereas various doses of IL-1 beta incubated with the cells for 12-72 hr showed no effect on the intracellular content of LH. Pretreatment with IL-1 beta for 48 hr inhibited both GnRH-mediated LH and FSH secretions by the pituitary. The secretion of FSH and LH mediated by an activator of protein kinase C, phorbol 12-myristate 13-acetate, was also significantly suppressed by pretreatment with IL-1 beta for 48 hr. These results suggest that (a) IL-1 beta has opposite effects on the secretion of LH and FSH and (b) pretreatment with IL-1 beta suppresses GnRH-mediated stimulation of LH and FSH by the pituitary and this suppressive effect of IL-1 beta may involve the suppression of a protein kinase C-dependent mechanism.

This study examined the uterine oxytocin- (OTR) and estrogen- (ER) receptor mRNA levels during and after pseudopregnancy (PSP) in rats. An increased OTR mRNA level was observed from day 14 of PSP, and the maximal level was attained during the following proestrus. The levels of ERalpha mRNA were low during PSP and significantly increased during the following estrus. The level of ERbeta mRNA was significantly decreased during proestrus and then returned to the values observed during days 7-14 of PSP by estrus. These results suggest i) suppression of ERalpha mRNA during the luteal phase and that ii) the changes in OTR, ERalpha and ERbeta mRNA levels during proestrus and estrus following PSP are similar to those during the normal estrous cycle.

The influence of ghrelin on feeding behaviour during infancy is unknown. To determine whether ghrelin influences milk intake in rat pups, newborn rats received a single i.p. injection of either rat ghrelin (100 microg/kg) or rabbit anti-ghrelin immunoglobulin G (100 microg/kg) every 5 days from postpartum day 5 to day 30 (P5-P30). Milk intake was then assessed by body weight gain following a 2-h suckling period. Ghrelin significantly increased weight gain relative to vehicle-injected controls in P20, P25 and P30 pups, but not in younger animals. Similarly, after 8 h of milk restriction, anti-ghrelin injections significantly decreased weight gain in P25 and P30, but not in younger pups. Interestingly, however, ghrelin did increase independent feeding in P10 and P15 pups using a paradigm in which pups consumed milk from a milk-soaked paper towel. We therefore conclude that ghrelin stimulates milk intake at an early postnatal stage, primarily by affecting adult-type feeding behaviour.

Bilateral lesions of the hypothalamic paraventricular nuclei (PVN) induce hyperphagia and obesity, and ghrelin stimulates appetite in rodents and humans. Conversely, corticotrophin-releasing hormone (CRH) and melanotan-II (MT-II, a synthetic structural homologue of alpha-melanocyte-stimulating hormone, alphaMSH) inhibit feeding behavior. The purpose of the present study was to determine whether these peptides are involved in the hyperphagia and obesity induced by PVN lesions. After bilateral electrolytic lesions of the PVN, rats were given ghrelin intraperitoneally (i. p.), or intracerebroventricular (i. c. v.) infusion of CRH or MT-II. We measured the cumulative food intake (FI) for 4 h after ghrelin injection in rats fed AD LIB, and the changes in FI at 15 min, 30 min, 1 h, and 2 h after infusion of CRH and MT-II in rats fasted for 24 h. Ghrelin significantly increased cumulative FI, with maximal response 3 h and 4 h after injection, and at these times, the FI of PVN-lesioned rats was greater than that of sham-operated rats. CRH significantly decreased FI in all experimental animals, but at 1 h, there was a more powerful inhibitory effect on FI in the PVN-lesioned group than in the sham-operated group. MT-II decreased FI in sham-operated, but not in PVN-lesioned rats. Thus, ghrelin and CRH showed more potent orexigenic and anorectic effects in PVN-lesioned rats, respectively, but MT-II lost its inhibitory action on feeding behavior. These results suggest that the hyperphagia and obesity induced by PVN lesions may be related to an increased orexigenic action of ghrelin due to the destruction of endogenous CRH and alphaMSH receptors.

Progesterone plays important roles in the regulation of female reproduction. In this study, progesterone receptor (PR) mRNA levels in rat uterus during pregnancy, labor, lactation and the estrous cycle were examined by competitive RT-PCR. During pregnancy and lactation, PR mRNA levels had decreased on day 20 of pregnancy (P20) and P21 compared with P15 but increased during labor. After a decline on day 1 of lactation (L1), PR mRNA levels had increased again on L3 and L14 compared with P15, P18, P20, P21 and P21pm (at 2200-2300 h on P21). There was no significant change in the PR mRNA level during the estrous cycle. The PR mRNA level did not change during 1 week of progesterone treatment or afterwards. Injection of 17beta-estradiol did not affect PR mRNA levels in rats treated with progesterone or those without any injections. In rats on P18, 17beta-estradiol injection did not change PR mRNA levels after sham-operation but induced an increase in PR mRNA levels of rats ovariectomized 6 h before the treatment. These results suggest that uterine PR mRNA levels are differently regulated during late pregnancy, labor and lactation, and during labor estrogen is one of the essential factors for the increase in PR mRNA levels.

Melanin-concentrating hormone (MCH) is believed to be an important orexigenic peptide mainly localized in the lateral hypothalamic area. Its involvement in the hyperphagia induced by hypothalamic lesions and lactation remains unclear. In this study, we investigated MCH immunoreactivity in the hypothalamus using immunohistochemistry and MCH concentration in the peripheral circulation using an enzyme immunoassay in rats with a lesion in the ventromedial hypothalamus or the paraventricular nucleus, and in lactating rats. Bilateral lesions of the ventromedial or paraventricular nuclei were performed using an electrolytic method. Quantification of immunoreactivity by image analysis revealed that the number and mean staining intensity of MCH-immunoreactive neurones in the lateral hypothalamic area and the zona incerta were significantly decreased by both types of lesions compared to sham controls, whereas circulating MCH concentration was not significantly different on day 7 postlesion. By contrast, in lactating rats on days 11-12 postpartum, the expression of MCH in the lateral hypothalamic area and the zona incerta was significantly increased compared to nonlactating controls. Circulating MCH concentration was not changed in lactating rats. These results suggest that hyperphagia induced by lactation, but not hypothalamic lesion, might be induced by excessive expression of MCH in the lateral hypothalamic area and the zona incerta.

The hypothalamic dorsomedial nucleus is suggested to be a final relay site for the afferent pathway of milk-ejection reflex. Existence of prolactin releasing peptide-immunoreactive cells in the dorsomedial nucleus and synaptic contact of prolactin releasing peptide-immunoreactive terminals with oxytocin cells was reported. Experiments were done to test the effect of prolactin releasing peptide on the electrical activity of oxytocin cells in the supraoptic nucleus. In rat brain slice preparations, oxytocin cells were unresponsive to the peptide. In lactating rats, although lateral ventricular injection of prolactin releasing peptide (20 nmol) was ineffective, a hundred nanomoles of the peptide increased basal activity and amplitude of milk-ejection related burst firing of oxytocin cells. Cells responded to lateral ventricular injection of peptides were unresponsive to direct application of peptides by pressure ejection from the recording electrode. These results suggest that prolactin releasing peptide may modulate electrical activity of oxytocin cells not through its direct action on oxytocin cells but through its action on area other than supraoptic nucleus.

Oxytocin receptor (OTR) mRNA levels increase dramatically near term and is potently stimulated by estrogen because increased OTR mRNA levels result from estrogen treatment in ovariectomized rat uterus. In this study, OTR, estrogen receptor (ER) alpha and ERbeta mRNA levels in the rat uterus during the estrous cycle were examined by quantitative RT-PCR. OTR mRNA levels during the estrous cycle began to increase on diestrus (P0.05, vs value on estrus), reached maximal increase both in the morning (1000-1130 hr) and afternoon (1600-1630 hr) on proestrus (P0.01, vs metestrus, diestrus and estrus) and then declined on estrus. In contrast ER alpha mRNA levels began to decrease on diestrus, reached statistical significance both in the morning and the afternoon on proestrus (P0.01, vs metestrus, diestrus and estrus) and returned to the value of metestrus on estrus. ERbeta mRNA levels were low in the morning and the afternoon on proestrus (P0.01, vs metestrus and estrus) and also returned to metestrus values on estrus. Treatments with estrogen for 3 days significantly decreased both ERalpha and ERbeta mRNA levels. It can be concluded from these results that during the estrous cycle, OTR mRNA levels in rat uterus predominantly increase at proestrus with a decrease in ERalpha and ERbeta mRNA levels, which is probably due to the increased estrogen levels in circulation before ovulation.

The widespread distribution of mRNA encoding orexin- 1 (OX1R) and -2 receptors (OX2R) in the central nervous system suggests that orexin may be involved in multiple functional pathways. Central administration of orexin stimulates feeding and also affects ovarian steroid-dependent luteinizing hormone secretion, suggesting involvement of orexin in the regulation of reproductive function. To investigate a possible role for orexin in reproductive function, we examined variations in prepro-OX, OX1R, and OX2R mRNA levels in the female rat hypothalamus during the estrous cycle, pregnancy, parturition, and lactation using competitive reverse transcription-polymerase chain reaction. During the estrous cycle, only OX1R mRNA expression during late proestrus was significantly higher than that at metestrus. The prepro-OX and OX1R mRNA levels on d 1 of lactation were significantly higher than that during late pregnancy and lactation. Immunohistochemistry revealed the presence of orexin-A immunoreactive cells and the OX1R subtype in the lateral hypothalamic area as well as the magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei, respectively, in pregnant and lactating rats. These results suggest a role for orexin in reproduction that may be involved in regulating physiological function in early lactation through important binding sites in hypothalamic PVN and SON.

The exact role of leptin in fasting has not been completely elucidated. To determine whether leptin can act in fasting to influence plasma estrogen levels and nitric oxide synthase reactivity in food regulating centers of the brain, we fasted female rats for 4 days and treated them i.p. with vehicle or 100 microg of recombinant mouse leptin as 1 ml on the 3rd and 4th day twice daily (10.00 and 17.00 h). Proestrus blood was collected at 10.00, 14.00, 18.00 and at 22.00 h, plasma obtained and assayed for estrogen (E2) and leptin levels. Verification of ovulation occurrence was by examining the oviduct for extruded ovum. The rat brains were removed and processed for nitric oxide synthase reactivity in the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) using NADPH-diaphorase histochemistry, a marker for neurons expressing NOS enzyme. Leptin effect on dependable variables such as food intake, water intake and body weight gain was also investigated. Four days fasting significantly decreased body weight, estrogen and postfast leptin levels, nitric oxide reactivity in the VMH and ARC nucleus and stopped ovulation in many (4 out of 5) rats fasted and given vehicle. Leptin treatment significantly increased plasma estrogen and postfast leptin levels, restored ovulation in many (4 out of 5) rats and increased nitric oxide reactivity in the VMH and ARC. Leptin significantly inhibited food intake, water intake and gain in body weight during recommenced feeding. These observations suggest that leptin could act in the pituitary-ovarian axis during fasting to improve reproductive function by partly stimulating estrogen secretion.

The present study was designed to investigate a possible role for ovarian steroids in the regulation of rat uterine oxytocin receptor (OTR) mRNA expression before labour. By using a competitive RT-PCR system, we have previously reported that parturition was associated with high levels of uterine OTR mRNA in all the animals examined. On the other hand, near term, some rats showed high OTR mRNA levels while others did not. We therefore examined the changes in OTR mRNA expression before and during prostaglandin F(2)(alpha) (PGF(2)(alpha))-induced parturition; a paradigm adopted to reduce the variation in the onset of parturition. Injection of PGF(2)(alpha) on day 18 of pregnancy significantly increased OTR mRNA expression in all the rats within 24 h of treatment, suggesting that the variation in OTR mRNA levels during spontaneous parturition may be due to the difference in the timing of the onset of parturition. The increase in OTR mRNA was significantly abolished by injection of the anti-oestrogen compound, tamoxifen. The stimulatory action of oestrogen on OTR mRNA expression was then examined in the presence or absence of ovarian factors. Pregnant rats were ovariectomized (OVX) or sham-operated on day 18 of pregnancy and either oestrogen or vehicle was administered 6 h after the surgical operation. Oestrogen increased OTR mRNA significantly in OVX rats 18 h after administration compared with sham-operated animals. Moreover, ovariectomy alone on day 18 of pregnancy increased OTR mRNA expression to a level which reached statistical significance 24 h after the operation. In addition, oestrogen treatment increased OTR mRNA levels in OVX virgin rats in which progesterone tubes were implanted for 1 week and removed 6 h before oestrogen injection. The stimulatory effect of oestrogen was not observed in rats in which the progesterone tubes were implanted for 1 week and not removed. These results suggest that the decline of progesterone is necessary for the expression of the stimulatory effects of oestrogen on uterine OTR mRNA.

In urethane-anaesthetized male rats, electrical stimulation of the dorsal penile nerve (DPN) excited 29 of 48 (60%) oxytocin cells in the contralateral supraoptic nucleus, whereas only 5 of 28 (18%) vasopressin cells were excited by the stimulation. The stimulus applied to the ipsilateral DPN to the recorded neurone also excited a similar proportion of oxytocin cells (25 of 43; 58%). Tactile stimulation of the glans penis excited 7 of 12 (58%) oxytocin cells, whereas the same stimulation excited only 3 of 16 (19%) vasopressin cells. The results suggest that sensory information arising from the penis preferentially excites oxytocin cells in the supraoptic nucleus.

Recent evidence suggests that endogenous nitric oxide (NO) contributes to the modulation of hormonal secretion from the anterior pituitary gland according to the physiological state of the animal. In this study, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and specific neurochemical assay were used to asses possible changes of NO synthase (NOS) activity in the anterior pituitary during pregnancy and parturition in rats. The anterior pituitary showed (weak) NADPH-d activity throughout pregnancy. Parturition increased the number and intensity of NADPH-d-positive cells. The NADPH-d-positive cells co-localized with immunofluorescent LH-positive cells. No variation in NADPH-d activity was apparent during the various stages of the oestrous cycle. Furthermore, NOS activity during parturition increased significantly when compared with non-pregnant and pregnant rats. Increases in both specific activity and NADPH-d activity gradually decreased within 24 h post-partum, suggesting that NO may modulate anterior pituitary function during parturition.

Bone inducing activity in demineralized bone matrix (DBM) of young spontaneously hypercholesterolemic (SHC) rats has been shown to be lower than that of aged SHC rats. This study examined the involvement of bone follistatin, an activin-binding protein, in bone induction. Immunoreactive follistatin was higher in DBM from 10-week-old SHC rats (DBM-10wk) than in DBM from 6-month-old SHC rats (DBM-6mo). When DBM without follistatin supplement was implanted, the C-propeptide of type II procollagen and calcium contents on day 12 in implants of DBM-6mo were 68% and 40% higher than those of DBM-10wk, respectively. In contrast, follistatin supplement to DBM decreased C-propeptide of type II procollagen and calcium contents in implants of both DBM-10wk and DBM-6mo, and the levels of these parameters were comparable between DBM-10wk and DBM-6mo, indicating reduced formation of cartilage and bone. These findings suggest that 1) follistatin content in bone matrix decreases with advancing age in SHC rats, and 2) the follistatin interferes with endochondral bone formation. We demonstrate that the lower bone induction of DBM from young SHC rats was partly due to the abundance of follistatin in bone matrix.

Large changes in the responsiveness of target organs to oxytocin are thought to originate from alteration of the number of oxytocin receptors (OTR). To elucidate the molecular mechanisms regulating the synthesis of the OTR, we developed a competitive reverse transcription-PCR protocol to measure OTR mRNA. We synthesized cRNA comprising a small stuffer introduced into the target mRNA. Using this cRNA as an internal standard, we made a quantitative estimation of OTR mRNA. Application of this method to the rat uterus revealed that the mean levels of OTR mRNA remained unchanged until 1030–1100 h on day 21 of pregnancy, increased significantly after 2200–2230 h on the same day and declined rapidly after parturition. A similar rapid increase in uterine OTR mRNA content was observed in rats given prostaglandin on day 18, inducing premature delivery on day 19 of pregnancy. All parturient rats had higher OTR mRNA levels regardless of whether parturition was spontaneous or prostaglandin induced. However, in a few rats, OTR mRNA remained as low as that observed during mid pregnancy even on day 22 of gestation, the expected day of parturition in about 70% of the rats in our colony. A similar increase in uterine OTR mRNA content to that observed at parturition was induced by oestrogen treatment for 3 days in ovariectomized virgin rats, but concomitant injection of progesterone did not influence the effect of oestrogen. The present results revealed that the large increase of uterine OTR at the peripartum period is accompanied by an increase in OTR mRNA content that may be brought about, at least in part, by increased oestrogen secretion following luteolysis. Journal of Endocrinology (1996) 150, 479–486

We examined the effect of nitric oxide (NO) on the process of parturition in rats. Subcutaneous injection of the NO donor sodium nitroprusside (SNP) in late pregnancy prolonged the total parturition time. The effect of N omega-nitro-L-arginine was not significantly different from that of saline. Intracerebroventricular injection of SNP in parturient rats delayed the progress of parturition. Both modes of SNP treatment also inhibited expression of maternal behavior. Central injection of oxytocin (OXT) with SNP failed to reduce parturition time significantly, but intrapartum, not postpartum, maternal behaviour was restored. These observations suggest that NO interferes with the release of OXT within the brain, hence affecting the initiation of maternal behaviour, and may also impair oxytocin secretion from the neurohypophysis.