Apoptotic Cell Death, bax and bcl-2 Expression During Sheep Mammary Gland Involution

Involution in ovine mammary tissue was studied by light and electron microscopy, and bax and bcl-2 protein distribution was examined by immunohistochemistry from the last day of lactation until the 8th day of drying off. The mammary gland alveoli were examined and the area of glandular epithelium was evaluated morphometrically. Regression of mammary gland epithelium by apoptosis was first identified 2 days after the end of lactation, and increased until day 8. Bax protein was detected throughout this period and was highest on the eighth day. A weak positive reaction for bcl-2 was observed only on days 1 and 8 after cessation of lactation. It is concluded that sheep mammary gland involution involves cell death by apoptosis and that bcl-2 gene family members are involved in the process.     

Course of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in mammary secretions of the goat during end-pregnancy and early lactation

The epidermal growth factor (EGF) plays a crucial role in mammogenesis in many species. In ruminants, studies are limited, as EGF does not occur in peripheral plasma and specific analytical systems do not exist. Therefore a heterologous radioimmunoassay based on rhEGF was set up to monitor EGF in mammary gland secretions from goats during end-pregnancy and early lactation. IGF-I was measured with an established radioimmunoassay. Samples were collected from 13 goats for 25 days ante-partum and 25 days post-partum. Mammary gland secretions were obtained ante-partum by removing a small amount of the udder secretions (control half) or milking (stimulated half). Post-partum normal milk samples were collected. Blood samples were drawn by jugular venipuncture for the same period. EGF was found to occur in different molecular weight forms in the mammary glands. For routine measurements these proteins were extracted with acetone and not further separated. IGF-I and EGF concentrations in mammary secretions and similarly IGF-I in blood were high ante-partum and decreased slightly towards birth. IGF-I but not EGF is found in the peripheral plasma. Whereas IGF-I concentrations in blood were quite constant post-partum, IGF-I and EGF dropped in mammary secretions close to the detection limits. The decrease was more pronounced in the stimulated half than in the control half. The data support a synergistic role for EGF and IGF-I for mammogenesis. Both factors are further influenced by the milking stimulus and thus the functional state of the udder.     

小鼠乳腺发育泌乳过程中L型氨基酸转运载体1的表达研究

为阐明L型氨基酸转运载体1（L-type amino acid transporter 1,LAT1）的表达与乳腺发育和泌乳功能之间的关系,采用荧光定量RT-PCR技术和激光共聚焦显微技术对青春期、妊娠期、泌乳期和退化期小鼠乳腺中LAT1及其辅因子4F2抗原重链（4F2hc）表达含量和部位的变化进行研究。结果表明,青春期乳腺导管发育缓慢,LAT1和4F2hc在导管上皮细胞膜、肌上皮细胞膜及乳腺脂肪细胞膜上均低水平表达;妊娠期导管上皮细胞增殖分化加速,LAT1和4F2hc在乳腺小叶导管上皮细胞膜基底侧表达,表达水平上调;泌乳期乳蛋白合成和分泌旺盛,LAT1和4F2hc在腺泡上皮细胞膜的基底侧表达,表达量达到峰值;退化期乳腺组织功能性结构消退,乳腺对氨基酸的需求降低,LAT1和4F2hc的表达下降。提示,LAT1/4F2hc是小鼠乳腺组织中转运氨基酸的载体形式,LAT1和4F2hc的表达变化与乳腺发育、泌乳、退化的生理过程中氨基酸的需要量相关。     

Involution and redevelopment of the mammary gland in goats. Histological-histochemical studies after a lactation time of 8-10 months

7 chamois-coloured mountain goats were used to investigate histologically and histochemically the processes of involution and redevelopment of the mammary gland after a lactation period of 8 to 10 months. Tissue specimens were obtained by incision biopsy at drying-off two months prepartum and afterwards in intervals of 8-16 days up to parturition. The findings of this investigation were compared with results of two former investigations, in which involution and proliferation of the gland were studied separately, thus precluding an overlapping of the two processes. At drying-off, after an 8 to 10 month-lactation, sections indicative of active lactation occurred concomitantly with sections at various stages of involution, as well as early stages of redeveloping alveoli. At 16 days post drying-off, the sections indicating lactation at first examination were in a stage of maximum involution and transformation. Thus, the time required for involution was reduced by half compared to drying-off at peak lactation. At 32 days post drying-off, stages of proliferation predominated with only a few involuting glands. Specimens obtained thereafter contained only redeveloping glands. The histological and histochemical differentiation between areas of involution and those of proliferation may present difficulties during the mid-portion of the dry period. There appears to be an association between the length of the time interval from drying-off to parturition and the rate of tissue transformation in the caprine mammary gland; the rate is increased when the duration of the dry period is reduced.     

Expression of cysteine sulfinate decarboxylase mRNA in rat mammary gland

Our previous report demonstrated that high concentration of taurine is present in rat milk for the first few days of lactation and plays an important role in the body growth of rat pups. In the present study, gene expression of rate-limiting enzyme for taurine biosynthesis, cysteine sulfinate decarboxylase (CSD) were examined in rat mammary gland. By RT-PCR, CSD mRNA was found to be expressed in rat mammary gland like that in the liver. The expression level of CSD mRNA in the mammary gland was higher in the earlier lactational stage (days 1 and 6 of lactation) than that in the later lactational stage (day 14). CSD mRNA expression in the mammary gland of non-pregnant rats was only a trace level. By in situ hybridization analysis, CSD mRNA was demonstrated in the epithelial cells of the mammary gland. These results suggest that high concentrations of taurine in the milk are at least partially resulted from de novo synthesis of taurine in mammary gland epithelial cells and that the expression pattern of CSD mRNA may be responsible for the changes in taurine levels in the milk during a lactational period.     

Characteristics of Rabbit Transgenic Mammary Gland Expressing Recombinant Human Factor VIII

The objective of this research was to compare (i) the content of milk protein and recombinant human factor VIII (rhFVIII) in the milk of transgenic and non-transgenic rabbit females at three lactations and (ii) histological structure, ultrastructural morphology and occurrence of apoptosis in rabbit transgenic and non-transgenic mammary gland during third lactation and involution. Significant differences (t_0.05) in milk protein content were found between transgenic and non-transgenic at all three lactations. The percentage of apoptotic cells was significantly higher (t_0.01) in non-transgenic ones compared with transgenic mammary gland tissues (6.5% versus 2.4%) taken at the involution stage. Morphometrical analysis of histological preparations at the involution stage detected a significantly higher (t_0.05) relative volume of lumen in transgenic animals compared with non-transgenic ones (60.00 versus 46.51%). Ultrastructural morphology of the transgenic mammary gland epithelium at the involution stage revealed an increased relative volume of protein globules (t_0.05); at the lactation stage, a significantly higher volume of mitochondria (13.8%) compared with the non-transgenic (9.8%) ones was observed. These results, although revealing differences in some parameters of ultrastructure and histology, indicate no harmful effect of the mouse whey acid protein-hFVIII transgene expression on the state of mammary gland of transgenic rabbit females.     

Expression and subcellular localization of efflux transporter ABCG2/BCRP in important tissue barriers of lactating dairy cows,sheep and goats

Expression of efflux transporter ABCG2/BCRP in tissues barriers has shown to be associated with altered pharmaco‐ and toxicokinetics of xenobiotics. Until now, little is known about the functional expression of this transporter in dairy animals. We therefore systematically examined the expression and subcellular localization of ABCG2/BCRP in small intestine, colon, lung, liver, kidney and mammary gland in lactating cows, sheep and goats. Carrier expression was investigated by RT‐PCR and Western blot analysis showing highest expression of ABCG2/BCRP in small intestine and mammary gland, high levels in liver and moderate amounts of protein in lung, colon and kidney. Regarding subcellular localization, BCRP was predominantly found at the apical plasma membrane of small intestine, colon, bronchial epithelium, bile ducts and overall in endothelial structures in all tested species. In the mammary gland, there was strong apical staining of the alveolar epithelial cells and most of the ducts in all dairy ruminants. We also detected significantly elevated protein expression in lactating mammary gland compared with nonlactating cows, sheep and goats. Our results contribute to the role of BCRP in cytoprotection and disposition in important tissue barriers and may have important implications for veterinary pharmacotherapy of dairy animals using drugs identified as BCRP substrates.     

Histochemical behavior of succinate dehydrogenase and lactate dehydrogenase as well as ribonucleic acid in the epithelium of lactic ducts and alveoli of cow udder]

The activities of succinate dehydrogenase (SDR) and lactate dehydrogenase (LDH) as well as the ribonucleic acid levels in the epithelium of both the lactic ducts and alveoli of lactic glands were examined in heifers, close to full term, lactating cows, some of them with subclinical mastitis lesions, and udder biopsies of two cows. The activities of SDH and LDH as well as RNA levels were pronounced in the glandular epithelium of alveoli and in the epithelium of the lesser lactic ducts. All three parameters were also pronounced but at lower levels in the cells of the apical stratum of the two-layer epithelium in the greater lactic ducts. Relationships were found to exist between the enzyme activities and RNA levels and the lactation cycle. Their patterns and manifestations were more or less typical of those areas of the lactic gland which were affected by mastitis.     

Secretory component and IgA expression by epithelial cells in sow mammary gland and mammary secretions

Secretory component (SC) and IgA expression of epithelial cells were studied in the mammary tissue and mammary secretions of sows. In mammary tissue, SC was not detected until day 105 of gestation. From the time of delivery (day 115) to the time of established lactation, the proportion of epithelial cells containing sc rose from 20 per cent to nearly 100 per cent. There was no IgA in alveolar epithelial cells until day 105 of gestation; on day 115, IgA positive epithelial cells were present in 10 per cent of the alveoli, which increased to 80 per cent during lactation. Epithelial cells represented more than 20 per cent of the total cells in colostrum, and predominated over leucocytes in milk. In colostrum, these epithelial cells (9 to 15 μm) showed weakly positive membrane, sc, contained cytoplasmic SC and had a limited capacity for in vitro proliferation. Ten per cent of epithelial cells contained intracytroplasmic IgA. In milk, the epithelial cells were larger (15 to 40 μm) with a higher expression of both membrane and intracytoplasmic sc; 66 per cent of these cells expressed intracytoplasmic IgA. These data showed that the capacity of mammary epithelium to process IgA to secretory IgA was complete at the end of mammary gland organisation, and established that the epithelial cells of milk contribute to the transfer of IgA to neonates.     

荷斯坦奶牛乳腺发育中的乳糖变化研究

目的:研究荷斯坦奶牛乳腺发育中乳糖的变化规律.方法:高效液相色谱法测定荷斯坦奶牛乳腺发育中乳糖的含量.结果:围产期,乳腺中才出现可检测到的乳糖;泌乳期乳糖含量增高,泌乳140d达到峰值;退化期乳腺乳糖含量迅速降低,退化30d含量很少.结论:不同生理时期,荷斯坦奶牛乳腺中的乳糖含量不同.     

Mammary-derived growth inhibitor in lactation and involution.

Presence of mammary-derived growth inhibitor (MDGI) in mammary tissue of lactating and involuted cows was investigated. Eighteen lactating, non-pregnant high-producing Holstein cows were randomly assigned to 3 experimental groups of 6 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group were slaughtered at 2-3 d, and the others at 4-8 d following sudden cessation of milking. Cessation of milking occurred at approximately 300 d in lactation. Western blot analysis revealed the presence of MDGI in the cytosolic and microsomal fractions of mammary tissue homogenates. High levels of MDGI were detected in mammary tissue obtained from lactating non-pregnant cows. A dramatic reduction in MDGI was observed in early involution (2-3 or 4-8 d following cessation of milking). These data suggest that a relationship exists between MDGI levels and the physiological status of the gland. Lack of MDGI may play a role during the processes of mammary involution and development prior to parturition.     

Gene expression and immunolocalization of fibroblast growth factor 2 in the ovary and its effect on the in vitro culture of caprine preantral ovarian follicles

This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro.     

Modulation of apical Na+/Pi cotransporter type IIb expression in epithelial cells of goat mammary glands

Recently, the type II sodium-dependent phosphate cotransporter NaPi IIb, which mediates the intestinal phosphate transport, was detected in the apical membranes of caprine mammary gland epithelial cells. Regulatory influences of developmental stages, dietary phosphorus (P) supply and hormones like calcitriol are well described for the intestinal NaPi IIb. Therefore, it was the aim of this study to examine the influence of involution and dietary P restriction on the expression of mammary gland NaPi IIb and of vitamin D receptor (VDR). During involution both, NaPi IIb and VDR, showed an initial increase of expression. This resulted in a delayed response on protein level. Dietary P restriction resulted in a decrease of mRNA expression which was not reflected on protein level. Influenced expression pattern, at least on mRNA level, indicate that mammary gland NaPi IIb is a regulated phosphate transporter which might have an important role especially during involution. Coexpression pattern with VDR provides an indication that calcitriol could be the modulator of these adaptive responses to involution and dietary P restriction. Therefore, a physiological meaning of NaPi IIb in mammary gland epithelia during processes of cell regeneration has to be considered.     

Preparation of an epithelium-free mammary fat pad and subsequent mammogenesis in ewes

The objective of this study was to develop a surgical procedure for the preparation of an epithelium-free mammary fat pad (cleared mammary fat pad; CFP) in ewes. At 7 to 10 d of age, ewe lambs (n = 43, mean BW 9.2 +/- .2 kg at 14 d) were sedated and one mammary gland was locally anesthetized. An incision circumscribing the base of the teat was made and blunt dissection was performed through the extraneous mammary fat pad tissue to enable the parenchyma and teat to be wholly removed. Failure to completely remove the epithelium enabled it to regenerate and grow into the mammary fat pad. Mean diameter of the parenchymal rudiment at 7 to 10 d of age was 8.9 +/- .5 mm (range of 5 to 16 mm). The excision site was closed with wound clips and recovered lambs returned to their dams. The contralateral mammary gland remained intact, allowing it to undergo normal development. Live weight gain was unaffected by this procedure. Ewes were subsequently slaughtered in groups at various stages of prepuberty, puberty, gestation, and lactation. Of 39 operated glands recovered, only one demonstrated epithelial outgrowth within the CFP. Parenchyma within the contralateral, intact gland underwent phases of rapid growth in prepuberty, puberty, and late gestation and was capable of milk synthesis after steroid induction or parturition. Change in weight of the CFP paralleled that of the intact mammary gland to 100 d of pregnancy. Sham CFP surgery was performed on four additional ewes wherein the parenchyma was completely excised and immediately replaced. Sham-operated epithelium populated the mammary fat pad and synthesized milk that could be expressed from the teat. A CFP in sheep will be a useful model for future investigations into the local growth regulatory mechanisms associated with ruminant mammogenesis.     

Aberrant development of mammary glands, but precocious expression of beta-casein in transgenic females ubiquitously expressing whey acidic protein transgene

It has been suggested that whey acidic protein (WAP) may function as a protease inhibitor. However, the actual function of WAP remains obscure. We investigated the histological development of the mammary glands of transgenic mice ubiquitously expressing WAP and CAG/WAP transgene. Ubiquitous expression of WAP induced aberrant development of the lobular alveoli of the mammary glands: mammary alveoli that were either aberrantly large or small in size increased in number in the developing mammary glands of these transgenic females during pregnancy and lactation. The expression of beta-casein was precociously induced in the mammary glands of the transgenic females during early pregnancy and accompanying this was a histological observation that abnormally developed lobular alveoli filled with milk proteins appeared in the mammary glands of transgenic females during early pregnancy. However, during lactation, the development of mammary glands was impaired in transgenic females. To investigate the possible paracrine action of WAP associated with mammary gland aberration, we transplanted the mammary tissue of CAG/EGFP transgenic females into the fat pad of virgin CAG/WAP transgenic females and initiated pregnancy by mating. The development of mammary tissue transplanted to the recipient was histologically examined on day 3 of lactation. The results revealed that the development of grafted mammary tissues was impaired in a manner similar to that of the mammary glands of CAG/WAP transgenic females, indicating that the inhibitory effect of WAP acts via a paracrine mechanism. In vitro experiments using HC11 cells with forced expression of exogenous WAP demonstrated the inhibitory function of WAP on proliferation of mammary epithelial cells.     

Expression of parathyroid hormone-related protein (PTHrP) mRNA in mammary gland of periparturient cows

The expression of parathyroid hormone-related protein (PTHrP) mRNA was examined in mammary gland with or without lactation, and during periparturient period in a Holstein cow and a Jersey cow. In the lactating mammary gland, PTHrP was detected in alveolar epithelial cells and the lumen by immunohistochemical analysis. The relative expression levels of PTHrP mRNA in mammary gland from lactating cows were significantly higher than those from non-lactating cows (P<0.05). During periparturient period, relative PTHrP mRNA level was remarkably low before the parturition in a Jersey and a Holstein cow, however, both levels were gradually increased and reached a peak level at 5-6 weeks after the parturition. In addition, the peak level in a Jersey cow was approximately 3-fold higher than that in a Holstein cow. From these results, PTHrP was synthesized and secreted in alveolar epithelial cells in mammary gland and increased subsequently with the lactation, suggesting a possible mechanism for the regulation of local calcium homeostasis.     

乳腺中脂肪细胞的转化和更迭

雌性动物的乳腺组织是由胚胎时期的外胚层发育而来,出生时只有少量导管和皮下基质结构,而后在性成熟、妊娠和泌乳期乳腺发育达到峰值,这一特点使乳腺成为出生后唯一可以重复再生的器官。在乳腺发育到退化的循环中,乳腺的上皮细胞、基质白色脂肪细胞、棕色脂肪细胞和肌上皮细胞经历了一系列转化和更迭,脂肪细胞的动态转化反映了乳腺的功能变化。研究乳腺细胞的转化和更迭与母畜的泌乳直接相关,对延续母畜的生产效率有重要意义。本文就乳腺中脂肪细胞转化方面的最新研究进展进行综述,为深度揭示乳腺发育过程中细胞更迭的机制提供前沿研究信息。     

Interleukin-8 expression by mammary gland endothelial and epithelial cells following experimental mastitis infection with E. coli

Epithelial and endothelial cells play a pivotal role in initiating and controlling the movement of leukocytes into tissues during inflammation through the production of cytokines and chemokines such as interleukin-8 (IL-8). In situ hybridization with an IL-8 riboprobe was used to determine IL-8 mRNA expression by mammary gland epithelial and endothelial cells in cows with experimental Escherichia coli mastitis. Epithelial cells of the gland, especially surrounding the alveoli, had increased IL-8 mRNA levels at all time points at which tissue samples were collected (8, 12, and 24h) after E. coli challenge. Levels of IL-8 expression in the epithelial cells decreased at 24h post-infection. IL-8 expression by mammary gland endothelial cells was low, but did increase slightly at 24h post-infection. Both epithelial and endothelial cells of the mammary gland can contribute to the production of IL-8 that is typically seen in coliform mastitis.