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Immunolocalization of Aquaporin Water Channels in the Domestic Cat Male Genital Tract
Full text linkFour different aquaporins (AQP1, 2, 5 and 9), integral
membrane water channels that facilitate rapid passive movement
of water, were immuno-localized in the excurrent ducts
collected from sexually mature cats during orchiectomy.
Aquaporins 1, 2 and 9, were immuno-localized at distinct
levels, whereas AQP5 was undetectable all along the feline
genital tract. No immunoreactivity was present at the level of
the rete testis with any of the antibodies tested. In the efferent
ducts, AQP1-immunoreactivity was strongly evidenced at the
apical surface of the non-ciliated cells, and AQP9-immunoreactivity
was shown at the periphery of both ciliated and nonciliated
cells. Aquaporins 2 was absent in the caput epididymidis,
either in the efferent ducts or in the epididymal duct.
Otherwise, AQP2 was increasingly localized at the adluminal
surface of principal cells from the corpus to the cauda
epididymidis and more weakly in the vas deferens epithelium.
The supranuclear zone of the epididymal principal cells was
AQP9-immunoreactive throughout the duct, with the exclusion
of the vacuolated sub-region of the caput and with higher
reaction intensity in the cauda region. AQP1 was present in the
blood vessels all along the genital tract. AQP1 was expressed
also in the smooth muscle layer of the vas deferens. The tested
AQP molecules showed a different expression pattern in
comparison with laboratory mammals, primates and the dog,
unique other carnivore species studied to date. The present
information is possibly useful in regard to the regional
morphology of the feline epididymis and correlated functions,
which are still a matter of debate
MORPHOLOGICAL FEATURES OF THE REPRODUCTIVE ORGANS IN DOMESTIC SPECIES IN RELATION TO WATER MOVEMENT CONTROL, HYPONUTRITION AND ENVIRONMENTAL INFLUENCES IN FETAL AND PREPUBERTAL LIFE, AND METABOLISM REGULATOR FACTORS.
Full text linkAQPs are a class of small, hydrophobic, integral membrane proteins that are able to make the cell membrane 10 to 100 fold more permeable than the membranes lacking such channels. Based on studies with AQP1, it appears that AQPs exist in the plasma membrane as homotetramers. Each AQP monomer contains 2 hemi-pores, which fold together to form a water channel. Different aqps have different patterns of glycosylation. In the case of AQP1 the peptide backbone is roughly 28 kDa and the glycosylated forms range from 40 to 60 kDa in mass.
Water transport activity of AQP1 is inhibited by mercurial ion, and the site of mercurial inhibition is the cysteine in the E loop. I have personally verified that using the bovine ovary granulosa cell swelling and shrinkage as a model for mercurial inhibition. The shortest pathway for water to reach the follicle antral cavity is the transcellular pathway directly through the cytoplasm of granulosa cells. In this first experiment we have considered the functional presence of Hg2+-sensitive Aqps in isolated granulosa cells assessing changes in cell volume in presence or absence of HgCl2.
Each of the AQPs has an essentially unique pattern of expression among tissues and during development. In humans 13 AQPs have been identified to date although this number pales in comparison to the greater than 100 related proteins found in plants and bacteria.
The landmark discovery of AQPs starts the era of molecular water channels, which has permeated virtually every branch of biology and medicine.
A number of recent studies have revealed the importance of mammalian AQPs in both physiology and pathophysiology and have suggested that pharmacological modulation of aquaporin expression and activity may provide new tools for the treatment of variety of human disorders, such as brain edema, glaucoma, tumour growth, fertility and obesity in which water and small solute transport may be involved.
Since multiple isoforms of AQPs are differentially expressed in cells and tissues, their localization to specific cells and membrane domains is critical to understanding the role of each AQP in the transfer of water and small solutes.
Balancement of water concentration within the uterine environment is important in every reproductive moment of a female mammal life. Rapid transcellular water movements are facilitated by AQPs that increase plasma membrane permeability. The results from my research showed that AQP-mediated water movement between the intraluminal, interstitial, and capillary compartments is crucial in the uterine imbibition mechanism in the bitch, including periodic stromal oedema in preparation for embryo implantation, with evidence that the expression of different AQPs can be regulated by steroid sex hormones. Also, the importance of the AQP-mediated fluid regulation in uterine environment in the peri-implantation period is known in the woman and laboratory mammals, to assure the successful implantation of blastocyst and its survival. The establishment of pregnancy and its maintenance is dependent on strict synchronization of uterine receptivity with embryonic maturation. Water homeostasis during foetal development is of crucial physiologic importance. Amniotic fluid (AF) provides the fluid-filled compartment that is essential for normal foetal growth, movement and development. Placental water flux is an important factor in determining AF volume and fetal hydration, too. AF changes during gestation may be dependent on modulations in solute as well as water permeability and changes in AQPs during gestation suggest their role in mediating placental and membrane water flow.
AQPs are channel proteins expressed in cell membranes of many epithelia involved in fluid transport and several AQPs have been identified in adult rat excurrent ducts, where considerable fluid reabsorption occurs. Concerning the morphological changes allowed by the AQP influence in the male reproductive tract I considered first of all the role of AQPs in the epididymis of a domestic species, the cat, and two animal models, the rat and the lamb.
Ultrastructural features of the epithelium lining the efferent ducts (ED) in the cat, as in other mammalian species, are strongly indicative of an absorptive activity taking place towards the intraluminal fluids. It is well-known that more than 95% of the fluid leaving the testis is reabsorbed by the ED, but the cell structures involved in the reabsorption processes are still a matter of debate. The purpose of the present work was to study the absorptive pathways in the ED of adult cats by means of 1) the immunohistochemical localization of different isoformes of the AQPs and 2) the localization and the carbohydrate characterization of the endocytotic apparatus by means of the lectin histochemistry. AQP-mediated trans-cellular route together with fluid phase glycocalix-mediated endocytosis.
Maternal undernutrition and, under certain circumstances, overnutrition, during pregnancy or during early postnatal life can alter reproductive function of the offspring. It is known that epididymal morphology in adult rats is affected by undernutrition during fetal to prepubertal life. For this study I tested the influence of pre- and postnatal undernutrition on AQP-expression in the adult male genital tract.
Exposure to environmental endocrine disrupting chemicals (EDCs) during pregnancy in animals and humans is associated with reduced sperm counts and increased incidences of testicular cancer and reproductive tract abnormalities. Although male fetuses from pregnant ewes exposed to EDCs contained in sewage sludge fertiliser (a “real life” exposure model) exhibit fewer testicular Sertoli and Leydig cells, the epididymis has not been investigated. The movement of fluids and small solutes in the epididymis is critical for adult sperm maturation and this process is regulated, in part, by the AQPs. I examined the effects of sewage sludge exposure on the expression of AQPs in the late gestation fetal epididymis.
The results I obtained suggest that exposure to environmental chemicals may affect the capacity of efferent ducts to transport water and small solutes thus influencing fluid resorption, protein synthesis and secretion. Ultimately, this may impact on sperm viability in the adult.
Another research item was the study of some factors which control the growth of the gubernaculum testis and regression of the cranial suspensory ligament during foetal development, resulting in transabdominal descent of the testes. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is supposed to induce the growth of the gubernaculum in male foetuses, thus being directly responsible for the testicular descent in humans and rodents. This is believed to happen with the LGR8 receptor intervention. A study led during my PhD provides evidence of the immunolocalization of INSL3 in the Leydig cells of dog male foetuses and of LGR8 receptor in different tissues of the gubernaculums testis of the same foetuses. AQPs are also involved in the water imbibition of gubernaculum testis: dog foetuses gubernacula showed a high expression of AQP1, AQP3, AQP4 and AQP9 respectively in blood vessels, smooth muscles, connective tissue and fat.
As well known AQPs are ubiquitarly distributed in the body maintaining all the same the specific function as channels to permeate water and small solutes. I collaborated with the University of Perugia regarding the immunolocalization of AQP5 in the sheep salivary glands in relation to the pasture vegetative cycle.
AQP5 is widely expressed in exocrine glands; in particular, in the rat salivary glands AQP5 is highly expressed in the apical plasma membrane of serous acinar cells, in the secretory canaliculi and in the intercalated duct cells, while it is lacking in mucous acinar cells and in striated ducts, indicating its important role in the saliva production (further supported by the observation of markedly depressed rates of salivary secretion in APQ5-deficient mice. I studied the presence of AQP5 in sheep parotid and mandibular glands, and its expression in the different stages of pasture vegetative cycle. AQP5 is expressed in parotid gland and the reactivity was higher when animals were fed on grasslands at the end of vegetative cycle than on grasslands at the maximum of vegetative development.
Ultimately I collaborated to a study created by the Reproduction Unit of the University of Milan regarding the relationship between nutrition and reproductive efficiency in the mare.
Leptin, the hormone product of ob gene expression, is an important endocrine indicator of adipose mass and nutritional status, as well as an important regulator of various aspects of feed intake, growth, metabolism and reproduction. Leptin amount in the blood is proportional to body energy stores and/or body mass, so, inadequate nutrition might impair reproductive function leading, for example, to the delayed onset of puberty. Indeed, the onset of puberty in humans and animals is associated with an increase in fat and consequent increase in circulating leptin, suggesting that leptin may be required for normal growth and development of reproductive organs. In the mare, besides many reports quantifying the correlation of circulating concentration of leptin with body condition scores1,2, only few informations exist about the presence of leptin (Ob) and leptin receptor (Ob-R) in the ovary or in the oocyte. The results support the hypothesis that in the horse leptin is differently localized during oocyte IVM showing different immunoreaction intensity related either to the horse breed or to the reproductive puberal development. The presence of leptin in the ovary could suggest a possible involvement in oocyte maturation, angiogenesis, follicle rupture or subsequent corpus luteum formation. Moreover, leptin may be involved in the determination of the animal pole of the oocyte and in the establishment of the inner cell mass and trophoblast in the embryo
Presence of Aquaporins in the bitch uterus and correlation with serum progesterone levels. Potential roles in uterine water movements during the oestrus cycle and the pregnancy
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Morphological evaluation of the placenta and fetal membranes during canine pregnancy from early implantation to term
No full textTo describe the histological changes of fetal adnexa throughout the physiological pregnancy, canine samples were obtained during natural delivery and caesarean section, as well as during ovariohysterectomy performed at any stage of undesired pregnancies (N=12). The first period of pregnancy (multiple samples collected at 10, 12, 14days) was consistent with pre- and peri-implantation events, i.e. apposition and initial invasion steps into the endometrium. The second period (multiple samples collected at 18, 38, 40, 45 days) was related to the development of extra-embryonic structures, placenta establishment and labyrinth formation. At the end of this period the maximum morphological complexity of the endotheliochorial placenta was achieved, characterized by complete erosion of the endometrial epithelium and underlying interstitium with exposure of maternal capillaries to the chorial cells. The third period of gestation (multiple samples collected at 50, 53, 57, 60, 63 days) was characterized by enhancement either of placental and extra-embryonic tissues
Functional evaluations of the cat epidydimis related to the expression of aquaporins and lectin-binding sites
No full textPrevious ultrastructural studies of the epithelium lining the cat epididymal duct
indicated that principal cells could be involved in fluid absorption as well as
glycoprotein secretion1. It is known that modifications of the surface glycoproteins
occur during the maturation, capacitation and acrosome reactions of spermatozoa, these
glycoconjugates most probably being of epithelial origin by a secretory process into the lumen2. Therefore, the purpose of the present work was to study the possible absorptive and
secretory events taking place in the cat epididymis by 1) the immunohistochemical
localization of four different isoforms of Aquaporins (AQP1, 2, 5 and 9), integral
membrane water channels that facilitate rapid passive movement of water, and 2) the
carbohydrate expression and localization in the epithelial lining, by means of lectin
histochemistry, utilizing a panel of 12 lectins in association with sialidase treatment, as
previously published3. The study was carried out on fragments of adult cat epididymides
obtained through orchiectomy, fixed in neutral formalin and paraffin-embedded.
AQP1-immunoreactivity, strongly evidenced at the apical surface of the ductuli
efferentes in non-ciliated cells, was present in the blood vessels all along the epididymis
but not in the lining epithelium. Otherwise, AQP2 was well localized at the adluminal
surface of principal cells in the cauda epidydimidis and AQP9 shows immunoreactivity
at the apical region of the principal cells throughout the duct. AQP5-immunoreactivity
was undetectable. Lectin histochemistry revealed the following binding sites. In
principal cells: 1) stereocilia showed greater reactivity with MAL II, PNA, s-PNA, s-
DBA, RCA120, WGA in the cauda and no change in the staining intensity with SNA,
HPA, SBA, Con A along the ductus; 2) the apical zone reacted with SBA in the whole
ductus and with SNA in the cauda; 3) the Golgi zone bound UEA I along the ductus,
whereas MAL II, DBA, RCA120, HPA, and WGA were bound in the caput and corpus
regions. Probable apical cells reacted with s-PNA and GSA I-B4 in the cauda
epididymis. Spermatozoa displayed a staining increase with MAL II, PNA, s-PNA, s-
DBA, RCA120, HPA, SBA, WGA moving to cauda epididymis but no change with SNA
and Con A. The regional differences in the lectin-binding pattern of the epithelium and
spermatozoa of cat epididymis could be related to the production of region-specific
glycoproteins implicated in the maturation and/or storage of spermatozoa.
Our data provide evidence for a possible pathway in the bulk reabsorption of water
throughout the cat epididymis. The abundant expression of AQP2 and AQP9 water
channels represents an important pathway for transmembrane water and solute
movement. Region-specific glycoproteins have also been evidenced, probably
implicated in the maturation and/or storage of spermatozoa
Preliminary data on the AQP5 immunodetection in sheep salivary glands related to the pasture vegetative cycle
No full textImmunolocalizzazione di INSL3 nelle cellule di Leyding fetali di cane e nel recettore LGR8 nel Gubernaculum testis
No full textImportanza delle aquaporine nell'equilibrio idrico dell'apparato riproduttore della cagna : bilanciamento dei fluidi oviduttali e possibile ruolo del meccanismo di imbibizione uterina
No full textIl movimento di fluidi attraverso le membrane cellulari è rilevante in
molti processi alla base della riproduzione. Il trasferimento passivo di acqua è facilitato
dalla presenza di Aquaporine, proteine canale di membrana, la cui espressione negli
organi riproduttori femminili muta con la fase funzionale. Lo studio immunoistochimico
di quattro diverse isoforme di AQPs nella salpinge di cagna mira a dimostrare il loro
coinvolgimento nelle funzioni secretorie e assorbenti atte a mantenere l’appropriato
milieu oviduttale, fornendo l’ambiente migliore per la fecondazione e le prime fasi dello
sviluppo embrionale. Anche la modulazione dell’imbibizione dei tessuti dell’utero
correlata all’andamento degli ormonali steroidei e in rapporto al possibile processo di
impianto embrionale, è rapportabile all’espressione di AQPs nei diversi tessuti
Presence of Aquaporins in the bitch uterus : is there a potential role in uterine water movements during different phases of the estrous cycle?
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