Decrease in the cytosolic NADP+-dependent isocitrate dehydrogenase activity through porcine sperm capacitation.

In order to understand the molecular mechanisms involved in the sperm capacitation, we have identified the proteins tyrosine-phosphorylated during the capacitation especially in conjunction with the regulation of the levels of reactive oxygen species (ROS) in sperm. In the present study, the effects of the tyrosine phosphorylation of cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) on its catalytic activity and on the levels of ROS in sperm have been studied. The tyrosine phosphorylated IDPc showed a significantly lowered enzymatic activity. The immunocytochemical analyses using the highly specific antisera against IDPc revealed that IDPc was mainly localized to the principal piece of the porcine sperm flagellum. As IDPc is one of the major NADPH regenerating enzymes in porcine sperm, it is strongly suggested that the decrease in IDPc activity is involved in the increased levels of ROS, which results in the induction of hyperactivated flagellar movement and capacitation.

CABCOCO1, a novel coiled-coil protein With calcium-binding activity, is localized in the sperm flagellum.

The gene 1700040L02Rik (GenBank accession number NM_028491, NP_082767.1) was selected by in silico screening as candidate that encodes a calcium-binding protein in sperm from a database of predicted mouse cilia-related genes. The predicted amino acid sequence revealed the presence of coiled-coil domain at the C-terminus and a CLAMP motif containing a leucine zipper domain in the middle of the protein. Assessment of a recombinant version of this protein by Stains-all and ruthenium red staining and by direct measurement of terbium binding revealed its calcium-binding activities. We therefore named this protein CABCOCO1 for calcium-binding coiled-coil protein-1. Immunohistochemical analyses showed its localization in spermatogenic cells of mouse testis. CABCOCO1 was first observed in the cytoplasm of murine spermatocytes, concentrated around centrioles of spermatids and co-localized with the centrosomal protein pericentrin. During the stage when centrosome number is reduced, CABCOCO1 relocalized to the murine sperm flagellum. On the other hand, in porcine sperm, whose proximal centriole remains intact while the distal centriole degenerates during spermiogenesis, CABCOCO1 localized both in the basal body and the flagellum. These results suggested that CABCOCO1 is involved in the control of sperm flagellar movement. Mol. Reprod. Dev. 83: 912-926, 2016 © 2016 Wiley Periodicals, Inc.

Porcine sperm capacitation involves tyrosine phosphorylation and activation of aldose reductase.

Mammalian sperm must be activated in the tubal isthmus through capacitation to induce the acrosome reaction and subsequent fertilization. Although the molecular mechanisms involved in capacitation have yet to be fully elucidated, increased concentrations of reactive oxygen species (ROS) and the extent of tyrosine phosphorylation of proteins have been suggested to play central roles in the completion of capacitation. In this study, aldose reductase was for the first time identified as one of the tyrosine-phosphorylated proteins involved in the capacitation of porcine cauda epididymal sperm. Both tyrosine phosphorylation and activity of aldose reductase associated with the particulate fraction of sperm cells were significantly enhanced during capacitation. Alrestatin, a membrane-permeable and specific inhibitor of aldose reductase, plays a role in the inhibition of aldose reductase activity, elevation of intracellular levels of ROS, and induction of hyperactivated motility, all at similar dose dependencies. Alrestatin canceled both the increase in the tyrosine phosphorylation of aldose reductase and the decrease in the glutathione levels in sperm-induced during capacitation. The hyperactivated motility was induced to a higher extent in the presence of glucose than in the presence of fructose. These results indicate that aldose reductase plays an important role in induction of hyperactivation and capacitation of sperm through the elevation of ROS in sperm cells. Furthermore, aldose reductase was shown to be added to sperm during transit through the epididymis, suggesting that aldose reductase is one of the key proteins that support the functional maturation of sperm.

Activated vitamin D3 and pro-activated vitamin D3 attenuate induction of permanent changes caused by neonatal estrogen exposure in the mouse vagina.

Exposure of mice to a high dose of estrogens including diethylstilbestrol (DES) during the neonatal period modifies the developmental plan of the genital tract, which leads to various permanent changes in physiology, morphology and gene expression. These changes include development of an abnormal vaginal epithelium lined with hyperplastic mucinous cells accompanied by Tff1 gene expression in mice. Here, the influence of vitamin D on the direct effect of estrogen on the developing mouse vagina was examined. The mid-vagina of neonatal mice was cultured in a serum-free medium containing estradiol-17ß (E2) and various concentrations of 1,25-dihydroxyvitamin D3 (1,25(OH)2D) ex vivo and then was transplanted under the renal capsule of ovariectomized host mice for 35 days. Exposure to E2 alone caused the vaginal tissue to develop estrogen-independent epithelial hyperplasia and to express TFF1 mRNA, while addition of a low nanomolar amount of 1,25(OH)2D added at the same time as E2 to the culture medium attenuated the effects of estrogen. Expression of vitamin D receptor was also evident in the neonatal mouse vagina. Interestingly, addition of 25-hydroxyvitamin D3, a pro-activated form of vitamin D, at the micromolar level was found to be potent in disrupting the developmental effects of E2, while cholecalciferol was not at least at the dose examined. Correspondingly, expression of Cyp27B1, a kidney-specific 25-hydroxyvitamin D hydroxylase, was evident in the neonatal mouse vagina when examined by RT-PCR. In addition, simultaneous administration of 1,25(OH)2D successfully attenuated DES-induced ovary-independent hyperplasia in the vagina in neonatal mice in vivo. Thus, manipulation of vitamin D influenced the harmful effects of estrogens on mouse vaginal development.

Localization of a novel RNA-binding protein, SKIV2L2, to the nucleus in the round spermatids of mice.

In our previous study (Kawashima et al., Biol Reprod 2009; 80: 1293-1304), we suggested that the first cycle of spermatogenesis recovered from busulfan-induced temporary arrest was a good model system to analyze the proteins expressed at the specific stages of spermatogenesis in the mouse, and this has been confirmed in the present paper. Namely, six-week-old mice were injected with busulfan at 20 mg/kg body weight. The germ cells except for the undifferentiated spermatogonia disappeared by 32 days after injection. The surviving spermatogonia started to proliferate, and spermatogenesis was entirely recovered about 77 days after injection. By proteome analysis of the busulfan-treated testis during the process of recovery of spermatogenesis, we identified a protein that was expected to be expressed in the spermatogenic cells as Superkiller viralicidic activity-2-like-2 (SKIV2L2). Skiv2l2 mRNA was found in the kidney, epididymis and heart as well as the testis. In the testis, Skiv2l2 mRNA was shown to be highly expressed in the spermatocytes at stages I to VI. On the other hand, SKIV2L2 protein was found to be predominantly localized in the nuclei of round spermatids. In accordance with the fact that SKIV2L2 belongs to the Ski2 family within the superfamily 2 of RNA helicases, it has been shown that SKIV2L2 has both the RNA-binding and ATPase activities.

Serotonin regulates mammary gland development via an autocrine-paracrine loop.

Mammary gland development is controlled by a dynamic interplay between endocrine hormones and locally produced factors. Biogenic monoamines (serotonin, dopamine, norepinephrine, and others) are an important class of bioregulatory molecules that have not been shown to participate in mammary development. Here we show that mammary glands stimulated by prolactin (PRL) express genes essential for serotonin biosynthesis (tryptophan hydroxylase [TPH] and aromatic amine decarboxylase). TPH mRNA was elevated during pregnancy and lactation, and serotonin was detected in the mammary epithelium and in milk. TPH was induced by PRL in mammosphere cultures and by milk stasis in nursing dams, suggesting that the gene is controlled by milk filling in the alveoli. Serotonin suppressed beta-casein gene expression and caused shrinkage of mammary alveoli. Conversely, TPH1 gene disruption or antiserotonergic drugs resulted in enhanced secretory features and alveolar dilation. Thus, autocrine-paracrine serotonin signaling is an important regulator of mammary homeostasis and early involution.

EFCAB2 is a novel calcium-binding protein in mouse testis and sperm.

Calcium-binding proteins regulate ion metabolism and the necessary signaling pathways for the maturational events of sperm. Our aim is to identify the novel calcium-binding proteins in testis. The gene EFCAB2 (GenBank NM_026626.3, NP_080902.1) was not previously examined, and its properties and exact mechanisms of action are unknown. In this study, we performed phylogenetic and structure prediction analyses of EFCAB2, which displays definitive structural features. Additionally, the distribution, localization, and calcium binding ability of mouse EFCAB2 were investigated. Results revealed extensive conservation of EFCAB2 among different eukaryotic orthologs. The constructed 3D model predicted that mouse EFCAB2 contains seven α-helices and two EF-hand motifs. The first EF-hand motif is located in N-terminal, while the second is located in C-terminal. By aligning the 3D structure of Ca2+-binding loops from EFCAB2 with calmodulin, we predicted six residues that might be involved in Ca2+ binding. The distribution of the Efcab2 mRNA, as determined by northern blotting, was detected only in the testis among mouse tissues. Native and recombinant EFCAB2 protein were detected by western blotting as one band at 20 kDa. In situ hybridization and immunohistochemical analyses showed its localization specifically in spermatogenic cells from primary spermatocytes to elongate spermatids within the seminiferous epithelium, but neither spermatogonia nor somatic cells were expressed. Moreover, EFCAB2 was specifically localized to the principal piece of cauda epididymal sperm flagellum. Furthermore, the analyses of purified recombinant EFCAB2 by Stains-all, ruthenium red staining, and by applying in vitro autoradiography assay showed that the physiological function of this protein is Ca2+ binding. These results suggested that EFCAB2 might be involved in the control of sperm flagellar movement. Altogether, here we describe about EFCAB2 as a novel calcium-binding protein in mouse testis and sperm.

Expression and function of cystine/glutamate transporter in neutrophils.

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS-mediated oxidant injury. Cystine/glutamate transporter, designated as system xc- and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian-cultured cells. In the present paper, we have investigated system xc- in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc- nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc- activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT-deficient (xCT-/-) mice had lower cysteine content than those from the wild-type mice. GSH levels in the xCT-/-cells decreased rapidly when they were cultured, whereas those in the wild-type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT-/-cells compared with the wild-type cells. These results suggest that system xc- plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.

Identification, localization, and functional analysis of the homologues of mouse CABS1 protein in porcine testis.

Previously, we have identified a calcium-binding protein that is specifically expressed in spermatids and localized to the flagella of the mature sperm in mouse, so-called mCABS1. However, the physiological roles of CABS1 in the male reproductive system have not been fully elucidated yet. In the current study, we aimed to localize and clarify the role of CABS1 in porcine (pCABS1). We determined for the first time the full nucleotides sequence of pCABS1 mRNA. pCABS1 protein was detected on SDS-PAGE gel as two bands at 75 kDa and 70 kDa in adult porcine testis, whereas one band at 70 kDa in epididymal sperm. pCABS1 immunoreactivity in seminiferous tubules was detected in the elongated spermatids, and that in the epididymal sperm was found in the acrosome as well as flagellum. The immunoreactivity of pCABS1 in the acrosomai region disappeared during acrosome reaction. We also identified that pCABS1 has a transmembrane domain using computational prediction of the amino acids sequence. The treatment of porcine capacitated sperm with anti-pCABS1 antiserum significantly decreased acrosome reactions. These results suggest that pCABS1 plays an important role in controlling calcium ion signaling during the acrosome reaction.

Increase in the number of integrinbeta1-immunoreactive monocyte-lineage cells in experimentally-induced adenomyosis in mice.

Uterine adenomyosis is a disease in which hyperplastic endometrial stroma and glands invade the myometrium. We have previously demonstrated that hyperprolactinemia leads to the development of adenomyosis in mice. In the present study, a subtracted cDNA library was made by suppression subtractive hybridization to find specific genes that are abundantly expressed in the adenomyotic but not normal tissue in mice. A cDNA fragment of integrinbeta1 (ibeta1) was found in the library, and the expression of the gene product was increased in the adenomyotic uteri at mRNA and protein levels. Intense ibeta1-immunoreactivity was localized on a group of cells dispersing throughout the endometrial stroma. The number of ibeta1-immunoreactive (ibeta1-ir) cells was significantly greater in the uteri of mice with adenomyosis than normal mice. The majority of the ibeta1-ir cells expressed CD14-ir signal, a marker for monocyte-lineage cells, whereas an increase in the number of CD14-ir cells was also evident in the adenomyotic uteri, especially in the ectopic endometrial tissue. Thus, the adenomyotic stromal tissue contained numerous monocyte-lineage cells with higher expression levels of ibeta1, one of their products. The relationship between the increased number of monocyte-lineage cells and the hyperplastic proliferation of endometrial tissues was discussed with a view to understanding the progressive mechanism of adenomyosis.

Priming effects of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 on the development of adenomyosis in the mouse uterus.

The possibility of therapeutic application of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 for preventing the development of uterine adenomyosis was investigated in mice. First priming effects of CP8816 on 17beta-estradiol (E2)-induced cell division in uterine tissues were examined. As a result, pretreatment with CP8816 or progesterone significantly suppressed the elevation of the mitotic activity in the luminal epithelial cells of mice treated with E2 later. Priming with CP8816 had little effect on the stromal cells, but progesterone priming caused an increase of stromal mitotic activity in mice treated with E2 later. To evaluate the inhibitory effect of these compounds on the development of adenomyosis induced experimentally by pituitary grafting, 7-week-old female mice were isografted with a single anterior pituitary in the uterus and divided into four groups. Two groups of mice were given daily subcutaneous injections of 1 mg of CP8816 or the vehicle alone for 6 weeks from the day after the grafting. Remaining two groups of mice were given oral administration of 1 mg of CP8863 or the vehicle only for 5 weeks starting one week after the grafting. The incidence of adenomyosis was significantly lower in the groups of mice treated with CP8816 and CP8863 than in the respective control groups. The mechanism by which CP compounds inhibited the development of adenomyosis might be related to their priming effects, i.e., their inhibitory effect on epithelial cell division and lack of effect on stromal cell division after subsequent exposure to E2.

Cortactin-binding protein 90 (CBP90) expression in the mouse mammary glands during prolactin-induced lobuloalveolar development.

We have previously performed suppression subtractive hybridization to identify genes that were induced during prolactin (PRL)-driven lobuloalveolar development of the mammary gland. This suggested that cortactin-binding protein 90 (CBP90), which is known to be a brain-specific protein that binds to cortactin, was expressed under the regulation of PRL in the mammary glands (preliminary observation). In this study, the expression of CBP90 was examined in the mammary glands of mice under manipulated hormonal circumstances. PRL treatment by 9 days of pituitary grafting induced CBP90 expression in the normal mammary glands but not in the cleared fat pads, while cortactin was expressed constitutively in both the normal mammary glands and the cleared fat pads. Unlike milk proteins, longer treatment with PRL (36 days of pituitary grafting) did not increase the expression level of CBP90 mRNA, while it slightly increased the cortactin mRNA level. Mammary CBP90 mRNA expression was induced by pituitary grafting but not by progesterone treatment in PRL-deficient mice, while pituitary grafting induced mammary CBP90 expression in ovariectomized PRL-deficient mice only when estrogen and progesterone were appropriately supplemented to permit the formation of alveolar buds. The CBP90 protein was detected by immunohistochemistry in the luminal epithelium of the alveolar buds and more faintly in the ductal epithelium. Thus, from the unique expression pattern, CBP90 may be useful as a molecular marker for the hormone-stimulated development of mammary alveolar buds.

Up-regulation of thymosin beta 4 gene expression in experimentally-induced uterine adenomyosis in mice.

Degradation of the myometrium contributes to the development of adenomyosis and disruption of the actin cytoskeleton occurs in the myometrium adjacent to the invasive endometrium. A subtractive cDNA library that contained genes up-regulated in the uterus with adenomyosis induced by pituitary grafting was constructed in mice. Thymosin beta 4 (Tb4), an actin sequestering protein, was contained in the library and was confirmed to be abundant in the adenomyotic uteri. Tb4-immunoreactivity was shown in the myometrium, with a stronger signal being observed in the area closer to the tip of endometrial tissue that is invading the myometrium. Up-regulation of Tb4 gene expression may be responsible for the disintegration of the musculature during the development of adenomyosis.

Water adaptation strategy in anuran amphibians: molecular diversity of aquaporin.

Most adult anuran amphibians except for the aquatic species absorb water across the ventral pelvic skin and reabsorb it from urine in the urinary bladder. Many terrestrial and arboreal species use a region in the posterior or pelvic region of the ventral skin that is specialized for rapid rehydration from shallow water sources or moist substrates. Periods of terrestrial activity can be prolonged by reabsorption of dilute urine from the urinary bladder. Aquaporin (AQP), a water channel protein, plays a fundamental role in these water absorption/reabsorption processes, which are regulated by antidiuretic hormone. Characterization of AQPs from various anurans revealed that the unique water homeostasis is basically mediated by two types of anuran-specific AQPs, i.e. ventral pelvic skin and urinary bladder type, respectively. The bladder-type AQP is further expressed in the pelvic skin of terrestrial and arboreal species, together with the pelvic skin-type AQP. In contrast, the pelvic skin-type AQP (AQP-x3) of the aquatic Xenopus has lost the ability of efficient protein production. The extra C-terminal tail in AQP-x3 consisting of 33 nucleotides within the coding region appears to participate in the posttranscriptional regulation of AQP-x3 gene expression by attenuating protein expression. The positive transcriptional regulation of bladder-type AQP in the pelvic skin and negative posttranscriptional regulation of pelvic skin-type AQP provide flexibility in the water regulation mechanisms, which might have contributed to the evolutionary adaptation of anurans to a wide variety of water environments.

CABS1 is a novel calcium-binding protein specifically expressed in elongate spermatids of mice.

Single intraperitoneal injection of busulfan at 20 mg/kg body weight to mature male mice induced the deletion of the spermatogenic cells, followed by the restoration of the spermatogenesis by the surviving undifferentiated spermatogonia. The changes of the protein contents in testis during these processes were analyzed by two-dimensional gel electrophoresis in order to identify the proteins expressed at the specific stages of spermatogenesis. An acidic protein that disappeared and recovered in the same time course as spermatids after the busulfan treatment was identified as CABS1 by mass spectrometry. It was found that CABS1 was specifically expressed in the elongate spermatids at steps 13 to 16 in stages I to VIII of the seminiferous epithelium cycle of the mouse, and then it localized to the principal piece of flagellum of the mature sperm in the cauda epididymis. We have found for the first time that CABS1 is a calcium-binding protein that binds calcium during the maturation in the epididymis.

Persistent trefoil factor 1 expression imprinted on mouse vaginal epithelium by neonatal estrogenization.

Exposure of female mice to estrogenic substances during the neonatal period induces developmental defects in the reproductive tract such as estrogen-independent persistent proliferation of the vaginal epithelium, which often leads to carcinogenesis in adulthood. In this study, several estrogen-regulated genes have been identified in the neonatal mouse vagina by DNA microarray hybridization analysis. Among the genes up-regulated in the developing vagina by a high dose of estrogen, trefoil factor 1 (TFF1), a mucin-associated gastrointestinal growth factor, showed a unique expression pattern in accordance with the irreversible changes induced by neonatal estrogenization in the vagina. Vaginal expression of TFF1 mRNA was markedly increased by estrogen in neonatal mice but not in adults, and pronouncedly intensified expression of the gastrointestinal gene was observed in the vagina of neonatally estrogenized mice even at adulthood. The specific localization of TFF1 protein in the epithelium of neonatally estrogenized vagina was confirmed by immunohistochemistry. Moreover, without any obvious alteration in the expression of gel-forming mucin genes, the lumen of the neonatally estrogenized vagina became filled with periodic-acid-Schiff-stained mucinous gel, which was possibly caused by the overexpression of TFF1. Thus, estrogen acts directly on the developing vagina in the permanent induction of TFF1 gene expression, and the gene induction does not appear to be related to hypermethylation of the cis-promoter of the TFF1 gene. TFF1 may be a useful marker for developmental estrogenization syndrome of the mouse vagina.

Neonatal estrogenization leads to increased expression of cellular retinol binding protein 2 in the mouse reproductive tract.

Exposure to estrogenic substances during a time window, the so-called "critical period," in perinatal life causes an irregular development of the genital tract that leads to ovary-independent proliferation and cornification in the vaginal epithelium in mice. We have previously demonstrated that retinol inhibits the irreversible effects of estrogen on the vagina. Here, mice kept in a vitamin-A-deficient condition during perinatal life were shown to be more sensitive to the harmful effects of estrogen. In addition, expression of mRNA for retinol binding protein type 2 (CRBP2), a "small intestine-specific" cytosolic protein that captures intracellular retinal and retinol, was detected in the vaginal epithelium. Induction of increased expression of CRBP2 mRNA by estrogen was also evident in the uterus and epididymis. Both estradiol-17beta and diethylstilbestrol markedly increased the tissue content of CRBP2 mRNA in the vagina and uterus during the neonatal "critical period" but not after 15 days of age. These results taken together imply that estrogen disrupts the local vitamin A balance by an induction of CRBP2 gene expression in the epithelium in the developing mouse genital tract, and that retinoid imbalance may contribute to the genesis of irreversible effects of estrogen on the vagina.

Involvement of keratinocyte growth factor (KGF)-KGF receptor signaling in developmental estrogenization syndrome of mouse vagina.

Exposure of mice to estrogen or keratinocyte growth factor (KGF) in vivo during the neonatal period results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. Here, whether and how KGF-signaling is involved in the effects of estrogen on the neonatal mouse vagina were studied with an in vitro method. Newborn mouse vaginae were cultured for 3 days in serum-free medium containing various combinations of estradiol-17beta (E2), KGF, anti-KGF antibody, KGFR inhibitory peptide and heparin, and then transplanted into ovariectomized host mice for 35 days. The vaginae cultured with 5 microg/ml E2 or 5 microg/ml KGF had a cornified thick epithelium, while the epithelium of the vehicle-treated controls stayed thin. The E2 effect was blocked by concurrent treatment with anti-KGF antibody or KGFR inhibitory peptide. KGF treatment alone at doses less than 500 ng/ml did not induce permanent vaginal changes but such changes did occur in vaginae treated with heparin plus as little as 10 ng/ml KGF. On the other hand, heparin inhibited the permanent vaginal changes induced by estrogen. These results suggest that irreversible vaginal changes are induced by the direct action of KGF on the developing vagina and that the developmental estrogenization syndrome of mouse vagina is caused by intensification of endogenous KGF/KGFR signaling by exogenous estrogen.

Vitamin A prevents the irreversible proliferation of vaginal epithelium induced by neonatal injection of keratinocyte growth factor in mice.

Exposure of female mice to estrogen during the perinatal period results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. However, the occurrence of such irreversible vaginal changes is blocked by concurrent vitamin A treatment. Neonatal exposure to keratinocyte growth factor (KGF), which is a paracrine mediator of epithelial-mesenchymal interactions, also induces the persistent proliferation and cornification of the vaginal epithelium in adult mice. This study was designed to examine whether concurrent administration of vitamin A inhibits the development of the irreversible vaginal changes in mice exposed neonatally to KGF. The vaginal epithelium in ovariectomized 35-day-old mice given 5 microg of KGF for 3 days after birth possessed a significantly larger number of layers and increased thickness as compared to that in control mice. Concurrent injections of 100 IU of vitamin A acetate inhibited the occurrence of the irreversible proliferation of the vaginal epithelium. These changes were equal to the results observed when 20 micro g of estrogen with or without vitamin A acetate was administered for 5 days after birth. Unlike the case of estrogen treatment, the effect of neonatal treatment with KGF seemed to appear after a latent period, since the vaginal epithelium did not show proliferation soon after the treatment. We discuss the inhibitory effect of VA on the irreversible vaginal changes induced by neonatal KGF treatment with reference to endocrine disruption by neonatal estrogen exposure.

Aldolase C/zebrin gene regulation by prolactin during pregnancy and lactation.

Prolactin (PRL) is necessary for the genesis of mammary alveolar buds and for lactation. A cDNA library enriched for PRL-dependent genes was made by suppression subtractive hybridization. Aldolase C/zebrin (AldC/zebrin), a brain-specific aldolase, was found to be PRL-dependent in the mouse mammary glands. AldC/zebrin was preferentially expressed in the alveolar buds. Expression of the gene in the ovary was also evident. During pregnancy, mammary AldC/zebrin mRNA levels were elevated beginning at midpregnancy (d 10 of pregnancy) in accordance with the genesis of the lobuloalveolar system, and the expression level was gradually increased through the end of pregnancy. Lactating mammary gland contained a very high level of AldC/zebrin mRNA, and the gene expression decreased during involution. By contrast, levels of aldolase A and B mRNA expression in the mammary glands were less affected by pregnancy and lactation. The selective regulation of AldC/zebrin may contribute to a shift in nutrient metabolism during pregnancy and lactation to facilitate epithelial growth and biosynthesis of milk constituents.