RESUMO
Cystic ovary disease (COD) is a common cause of subfertility in dairy cattle. Therefore, the aim of this study was to provide novel concepts for cyst classification and to investigate the effects of COD on tubal microarchitecture, oviductal metabolic function, and the formation of the sperm reservoir. Bovine Fallopian tubes affected by follicular cysts, follicular cysts with luteinization and luteal cysts were investigated by a variety of microscopic and histological techniques and compared to control cows in metestrus and diestrus. We defined three types of cysts involved in COD, each of which had a characteristic wall thickness, inner wall appearance and cellular pattern within the cyst aspirate. Regarding the Fallopian tube, each cyst type was associated with a characteristic morphology, specifically the microarchitecture of the folds in ampulla, epithelial cell ratios, and ciliated/secretory cell size and form. Furthermore, each cyst type showed different patterns of tubal glycoprotein and acidic mucopolysaccharide synthesis, which was highly variable as compared to the controls. Our studies are the first to characterize the effects of COD on the Fallopian tube, which promotes the establishment of novel, cyst-specific therapeutic concepts in cattle and helps gain a holistic view of the causes of subfertility in cows with COD.
Assuntos
Infertilidade , Cistos Ovarianos , Masculino , Feminino , Humanos , Bovinos , Animais , Tubas Uterinas/metabolismo , Sêmen/metabolismo , Cistos Ovarianos/veterinária , Cistos Ovarianos/metabolismoRESUMO
BACKGROUND: Although millions or even billions of sperm are deposited in the female genital tract, only very few sperm reach the oocyte, and only one single spermatozoon will successfully fertilize. During the journey of the sperm within the female genital tract, the interactions between spermatozoa and fallopian tube are critical for sperm selection, sperm survival, and maintenance of sperm fertilizing capacity. RESULTS: This review will provide a comprehensive overview of the latest findings regarding sperm transport and behavior of sperm within the oviduct, sperm selection in the oviduct, the formation of the sperm reservoir, and the release of sperm in the presence of the oocyte. It will primarily focus on recent novel insights on sperm-oviduct interactions, which have been obtained by cutting-edge technologies under in vivo or near in vivo conditions. CONCLUSIONS: The comprehensive analysis of the findings to date will elucidate the complex molecular changes in the tubal epithelium, which are induced by the presence of the sperm and will highlight how the epithelial cells of this organ affect transport, behavior, and function of sperm. This knowledge is essential for scientists and clinicians involved in assisted reproductive technologies.
Assuntos
Tubas Uterinas , Sêmen , Animais , Feminino , Fertilização/fisiologia , Humanos , Masculino , Oviductos , Espermatozoides/fisiologiaRESUMO
In the three decades since its inception, the sex-sorting technology has progressed significantly. However, field studies report conflicting findings regarding reproductive outcomes. Therefore, we conducted this meta-analysis of all trials published between 1999 and 2021. Non-return rates after 24 or 60 d (NRR 24/60), pregnancy, calving, abortion, and stillbirth rates were compared after AI with sex-sorted vs non-sorted sperm. Additionally, the impact of recent developments in the sex-sorting technology was assessed. Of 860 studies found, 45 studies (72 trials) provided extractable data and were included. Overall, the results of this meta-analysis provided evidence that the NRR 24/60 was diminished by 13%, pregnancy rates were reduced by 23% (25% cows, 21% heifers) and calving rates were reduced by 24% when using sex-sorted sperm. Enhancing the dosage to 4 million sex-sorted sperm/straw (including recent improvements, high vs low dose) as well as using fresh sex-sorted sperm (sorted vs non-sorted) increased pregnancy rate ratios by 7 percentage points. The refinement of the sex-sorting technology after 2015 resulted in a lowered reduction of pregnancy and calving rate of 19% and 23%, respectively. Whereas abortion rates were similar, the stillbirth of male calves was increased by 6.3%.
Assuntos
Criação de Animais Domésticos/métodos , Biotecnologia/métodos , Criopreservação/veterinária , Biologia do Desenvolvimento/métodos , Inseminação Artificial/veterinária , Prenhez , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Bovinos , Feminino , Fertilidade , Congelamento , Lactação , Masculino , Gravidez , Taxa de Gravidez , SêmenRESUMO
Cystic ovary disease (COD) is a common cause of bovine infertility but the impact of this disease on the oviduct is unknown. The aim of this study was to analyze the effects of COD on particle transport speed (PTS), ciliary beat frequency, myosalpinx contraction, and epithelial ion transport. Oviducts were obtained from cows affected by COD and compared with those of healthy, mid-diestrus cows. PTS and CBF were examined using live-cell imaging. Smooth muscle contraction and epithelial ion transport were investigated using organ baths and Ussing chambers. Our results showed that muscarinic receptors are involved in cholinergic signaling in the oviduct and that forskolin-induced cyclic AMP production is involved in active ion transport in the oviductal epithelium. Oviducts from cows with luteal cysts revealed significantly decreased PTS (p = 0.02). Further to that, in the oviducts of COD cows, the cholinergic regulation of smooth muscle contractions and active epithelial ion transport were significantly diminished (p < 0.0001). These results imply that in COD cows, oviductal transport is compromised by decreased fluid flow speed and reduced cholinergic regulation of smooth muscle contraction and ion transport. This knowledge contributes to a more comprehensive understanding of COD supporting the development of novel therapeutic concepts for infertility treatment.
Assuntos
Transporte de Íons/fisiologia , Contração Muscular/fisiologia , Cistos Ovarianos/veterinária , Oviductos/metabolismo , Animais , Bovinos , AMP Cíclico/metabolismo , Epitélio/metabolismo , Feminino , Cistos Ovarianos/metabolismo , Cistos Ovarianos/patologia , Oviductos/patologiaRESUMO
To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.
Assuntos
Oviductos/fisiologia , Capacitação Espermática/fisiologia , Espermatozoides/fisiologia , Animais , Bovinos , Tubas Uterinas/metabolismo , Feminino , Masculino , Microscopia Eletrônica de Transmissão/métodos , Oviductos/metabolismo , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismoRESUMO
An intact embryo-maternal communication is critical for the establishment of a successful pregnancy. To date, a huge number of studies have been performed describing the complex process of embryo-maternal signaling within the uterus. However, recent studies indicate that the early embryo communicates with the oviductal cells shortly after fertilizationand that this is important for the successful establishment of pregnancy. Only if the early embryo is capable to signal the mother within a precise timeframe and to garner a response, will the embryo be able to survive and reach the uterus. This review will give an overview of all the experimental designs which have investigated embryo-maternal interaction in the oviduct. In addition to that, it will provide a comprehensive analysis of the findings to date elucidating the morphological and molecular changes in the oviduct which are induced by the presence of the early embryo highlighting how the tubal responses affect embryo development and survival.
Assuntos
Comunicação Celular/fisiologia , Embrião de Mamíferos/fisiologia , Troca Materno-Fetal/fisiologia , Oviductos/fisiologia , Animais , Implantação do Embrião/fisiologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Tubas Uterinas/fisiologia , Feminino , Viabilidade Fetal/fisiologia , Humanos , Oviductos/citologia , GravidezRESUMO
Excess testosterone levels affect up to 20% of the female population worldwide and are a key component in the pathogenesis of polycystic ovary syndrome. However, little is known about how excess testosterone affects the function of the human fallopian tube-the site of gamete transport, fertilization, and early embryogenesis. Therefore, this study aimed to characterize alterations caused by long-term exposure to male testosterone levels. For this purpose, the Fallopian tubes of nine female-to-male transsexuals, who had been undergoing testosterone treatment for 1-3 years, were compared with the tubes of 19 cycling patients. In the ampulla, testosterone treatment resulted in extensive luminal accumulations of secretions and cell debris which caused ciliary clumping and luminal blockage. Additionally, the percentage of ciliated cells in the ampulla was significantly increased. Transsexual patients, who had had sexual intercourse before surgery, showed spermatozoa trapped in mucus. Finally, in the isthmus complete luminal collapse occurred. Our results imply that fertility in women with elevated levels of testosterone is altered by tubal luminal obstruction resulting in impaired gamete transport and survival.
Assuntos
Tubas Uterinas/efeitos dos fármacos , Fertilidade/efeitos dos fármacos , Testosterona/sangue , Testosterona/farmacologia , Pessoas Transgênero , Adulto , Muco do Colo Uterino , Cílios/enzimologia , Cílios/patologia , Doenças das Tubas Uterinas/induzido quimicamente , Feminino , Humanos , Histerectomia , Masculino , Pessoa de Meia-Idade , Ovariectomia , Síndrome do Ovário Policístico/patologia , Receptores Androgênicos/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/patologia , Testosterona/administração & dosagem , Testosterona/efeitos adversos , Adulto JovemRESUMO
Salpingitis is a common cause for subfertility and infertility both in humans and animals. However, the effects of salpingitis on tubal function and reproductive success are largely unknown. Therefore we set out to investigate the effects of inflammation on sperm and oocyte transport and gameto-maternal interaction in the oviduct using the bovine as a model. For this purpose, oviducts revealing mild (n = 45), moderate (n = 55) and severe (n = 45) inflammation were obtained from cows immediately after slaughter and investigated by live cell imaging, histochemistry and scanning electron microscopy. Our studies showed that endometritis was always correlated with salpingitis. Moderate and severe inflammation caused a significant increase in the thickness of tubal folds (p < 0.05). Severe inflammation was characterized by luminal accumulations of mucus and glycoproteins, increased apoptosis, loss of tight junctions and shedding of tubal epithelial cells. The mean ciliary beat frequency (CBF) in the ampulla was significantly reduced as compared to the controls (p < 0.05). The higher the grade of inflammation, the lower was the CBF (p < 0.001). In severe inflammation, spermatozoa were stuck in mucus resulting in decreased sperm motility. Our results imply that tubal inflammation impairs proper tubal function and leads to reduced sperm fertilizing capacity.
Assuntos
Endometrite/fisiopatologia , Células Epiteliais/metabolismo , Tubas Uterinas/fisiologia , Infertilidade/fisiopatologia , Oviductos/fisiologia , Salpingite/fisiopatologia , Espermatozoides/fisiologia , Animais , Apoptose , Bovinos , Endometrite/patologia , Células Epiteliais/patologia , Tubas Uterinas/patologia , Feminino , Fertilização , Glicoproteínas/metabolismo , Humanos , Infertilidade/patologia , Masculino , Muco/metabolismo , Salpingite/patologia , Motilidade dos Espermatozoides , Junções Íntimas/metabolismoRESUMO
In a recent genome-wide association study, 40 Fleckvieh bulls with exceptionally poor fertility were found to be homozygous for a nonsense mutation in the transmembrane protein 95 (TMEM95) encoding gene. Ejaculates from these individuals exhibited normal sperm concentration, morphology, viability, and motility. However, only 1.7% of inseminations resulted in pregnancies. The aim of this study was to examine the effect of this mutation in TMEM95 on bovine sperm function in vitro. Sperm from homozygous (mt/mt) males had lower in vitro fertility than sperm from wild-type (wt/wt) or heterozygous (wt/mt) bulls (P < 0.01). In addition, early embryo division was affected in the mt/mt group (P < 0.01). This translated into a lower (P < 0.01) blastocyst rate at day 8. Fluorescent staining revealed that TMEM95 is lost after the acrosome reaction. This led us to hypothesize that TMEM95 might be involved in events that lead to sperm-oocyte interaction. After fertilization, a lower number (P < 0.01) of sperm from mt/mt bulls bound to the zona pellucida (ZP). Sperm from mt/mt bulls were also less able to penetrate oocytes with no ZP (P< 0.01). However, when sperm from these animals were injected into mouse oocytes, they could decondense as successfully as sperm from wt/wt bulls. No differences between genotypes were observed in the ability of sperm to retain motility in an ex vivo oviduct, or in the percentage of sperm exhibiting markers for capacitation and acrosomal reaction. These results suggest that fertilization failure in mt/mt bulls is due to the inability of their sperm to interact with the oocyte vestments.
Assuntos
Doenças dos Bovinos/genética , Bovinos/genética , Infertilidade Masculina/genética , Proteínas de Membrana/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologia , Animais , Feminino , Masculino , Proteínas de Membrana/genética , Mutação , Interações Espermatozoide-Óvulo/genéticaRESUMO
Elevated estradiol levels are correlated with male infertility. Causes of hyperestrogenism include diseases of the adrenal cortex, testis or medications affecting the hypothalamus-pituitary-gonadal axis. The aim of our study was to elucidate the effects of estradiol treatment on testicular cellular morphology and function, with reference to the treatment regimen received. Testes samples (n = 9) were obtained post-orchiectomy from male-to-female transsexuals within the age range of 26-52 years. Each patient had a minimum of 1-6 years estradiol treatment. For comparison, additional samples were obtained from microscopically unaltered testicular tissue surrounding tumors (n = 7). The tissues obtained were investigated by stereomicroscopy, histochemistry, scanning electron microscopy (SEM) and immunohistochemistry. Our studies revealed that estradiol treatment significantly decreased the diameter of the seminiferous tubules (p < 0.05) and induced fatty degeneration in the surrounding connective tissue. An increase in collagen fiber synthesis in the extracellular matrix (ECM) surrounding the seminiferous tubules was also induced. Spermatogenesis was impaired resulting in mainly spermatogonia being present. Sertoli cells revealed diminished expression of estrogen receptor alpha (ERα). Both Sertoli and Leydig cells showed morphological alterations and glycoprotein accumulations. These results demonstrate that increased estradiol levels drastically impact the human testis.
Assuntos
Estradiol/farmacologia , Testículo/efeitos dos fármacos , Adulto , Colágeno/metabolismo , Receptor alfa de Estrogênio/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Glicoproteínas/metabolismo , Humanos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Espermatogênese/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia , Transexualidade/patologiaRESUMO
Nanocomposites with polypropylene as matrix material and nanoclay as filler were produced in a double twin screw extruder. The extrusion was monitored with a spectrometer in the visible and near-infrared spectral region with a diode array spectrometer. Two probes were installed at the end at the extruder die and the transmission spectra were measured during the extrusion. After measuring the transmission spectra and converting into turbidity units, the particle distribution density was calculated via numerical linear equation system. The distribution density function shows either a bimodal or mono modal shape in dependence of the processing parameters like screw speed, dosage, and concentration of the nanoclays. The method was verified with SEM measurements which yield comparable results. The method is suitable for industrial in-line processing monitoring of particle radii and dispersion process, respectively.
RESUMO
Routine infertility investigations in the male and female include imaging techniques such as ultrasonography and endoscopy (fertiloscopy). However, these techniques lack the resolution to localize vital sperm or to reveal detailed morphological analysis of the oviduct which is often the cause of infertility in females. Therefore we set out to evaluate the efficiency of optical coherence tomography (OCT) as a diagnostic imaging tool for micron-scale visualization of the male and female genital tract. Using the bovine as a model, the optical features of the Telesto(TM) , Ganymede(TM) (both Thorlabs) and Niris(TM) (Imalux) OCT imaging systems were compared.
Assuntos
Reprodução , Tomografia de Coerência Óptica/métodos , Animais , Bovinos , Feminino , Genitália Feminina/citologia , Genitália Masculina/citologia , Masculino , Medicina ReprodutivaRESUMO
The aim of this study was to assess the potential of probe-based confocal laser endomicroscopy (pCLE) as a new diagnostic imaging technique for the male genital tract. For this purpose, testes, epididymides, and vasa deferentia were obtained during transsexual surgery of healthy patients (n = 10, 26-52 years). Prior to this, testes of rats (n = 10, Sprague-Dawley) and mice (n = 8, wild-type) were examined. Ex vivo tissues were investigated by pCLE after topical fluorescence staining. Images and pCLE real-time video sequences were compared to images acquired by confocal laser scanning microscopy (CLSM); this allowed the identifying of corresponding microstructures. Interestingly, the seminiferous tubules of transsexual humans contained mainly spermatogonia due to long-term estrogen treatment, whereas the seminiferous tubules of the murine and rat spermatogenesis-related cell types were differentiated. Mosaicking improved the inspection potential by wide-angle views. Similarly, the microarchitecture of the epididymis and the vas deferens was successfully visualized in situ and on a cellular level by pCLE. In summary, pCLE allows for real-time identification of relevant microstructures responsible for spermatogenesis under ex vivo conditions. Additionally, pCLE enabled to localize vital spermatozoa in the testis thus opening up new ways to improve sperm retrieval rates during assisted reproduction. Both clinically relevant experiences hold promise to introduce this diagnostic method into a clinical study, and to investigate its potential as a clinical diagnostic procedure to expedite and improve the medical situation.
Assuntos
Genitália Masculina/citologia , Microscopia Confocal/métodos , Adulto , Animais , Genitália Masculina/fisiologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Ratos , Ratos Sprague-DawleyRESUMO
In azoospermic patients, spermatozoa are routinely obtained by testicular sperm extraction (TESE). However, success rates of this technique are moderate, because the site of excision of testicular tissue is determined arbitrarily. Therefore the aim of this study was to establish probe-based laser endomicroscopy (pCLE) a noval biomedical imaging technique, which provides the opportunity of non-invasive, real-time visualisation of tissue at histological resolution. Using pCLE we clearly visualized longitudinal and horizontal views of the tubuli seminiferi contorti and localized vital spermatozoa. Obtained images and real-time videos were subsequently compared with confocal laser scanning microscopy (CLSM) of spermatozoa and tissues, respectively. Comparative visualization of single native Confocal laser scanning microscopy (CLSM, left) and probe-based laser endomicroscopy (pCLE, right) using Pro Flex(TM) UltraMini O after staining with acriflavine.
Assuntos
Lasers , Imagem Molecular/métodos , Espermatozoides/citologia , Adulto , Ejaculação , Estudos de Viabilidade , Humanos , Masculino , Pessoa de Meia-Idade , Imagem Molecular/instrumentação , Fibras Ópticas , Testículo/citologiaRESUMO
Genetic variants underlying reduced male reproductive performance have been identified in humans and model organisms, most of them compromising semen quality. Occasionally, male fertility is severely compromised although semen analysis remains without any apparent pathological findings (i.e., idiopathic subfertility). Artificial insemination (AI) in most cattle populations requires close examination of all ejaculates before insemination. Although anomalous ejaculates are rejected, insemination success varies considerably among AI bulls. In an attempt to identify genetic causes of such variation, we undertook a genome-wide association study (GWAS). Imputed genotypes of 652,856 SNPs were available for 7962 AI bulls of the Fleckvieh (FV) population. Male reproductive ability (MRA) was assessed based on 15.3 million artificial inseminations. The GWAS uncovered a strong association signal on bovine chromosome 19 (P = 4.08 × 10(-59)). Subsequent autozygosity mapping revealed a common 1386 kb segment of extended homozygosity in 40 bulls with exceptionally poor reproductive performance. Only 1.7% of 35,671 inseminations with semen samples of those bulls were successful. None of the bulls with normal reproductive performance was homozygous, indicating recessive inheritance. Exploiting whole-genome re-sequencing data of 43 animals revealed a candidate causal nonsense mutation (rs378652941, c.483C>A, p.Cys161X) in the transmembrane protein 95 encoding gene TMEM95 which was subsequently validated in 1990 AI bulls. Immunohistochemical investigations evidenced that TMEM95 is located at the surface of spermatozoa of fertile animals whereas it is absent in spermatozoa of subfertile animals. These findings imply that integrity of TMEM95 is required for an undisturbed fertilisation. Our results demonstrate that deficiency of TMEM95 severely compromises male reproductive performance in cattle and reveal for the first time a phenotypic effect associated with genomic variation in TMEM95.
Assuntos
Estudo de Associação Genômica Ampla , Infertilidade Masculina/genética , Proteínas de Membrana/genética , Animais , Bovinos , Códon sem Sentido/genética , Humanos , Infertilidade Masculina/patologia , Inseminação Artificial/métodos , Inseminação Artificial/veterinária , Masculino , Espermatozoides/patologiaRESUMO
AIMS: In the oviduct, muscarinic acetylcholine receptors (MR) are linked with motility regulation and nicotinic receptors (nAChR) with ectopic pregnancy. We here aimed to determine the repertoire of cholinergic receptor expression in the murine oviduct and their functional coupling to regulation of intracellular calcium concentration ([Ca(2+)](i)). MAIN METHODS: Cholinergic receptor transcripts were assessed by RT-PCR in oviductal segments (ampulla, isthmus, uterotubar junction) in all cyclic stages and pregnancy, and in laser-microdissected samples of epithelium and smooth muscle, nAChR subunit α3 distribution in tissue sections using an appropriate genetic reporter mouse strain. [Ca(2+)](i) responses were monitored in ciliated and non-ciliated oviductal cells isolated from wild-type and MR subtypes 1 and 3 gene deficient mice. KEY FINDINGS: Transcripts for all MR subtypes (M1-M5) are constantly expressed whereas there is some variability in nAChR expression from individual to individual. The qualitative expression pattern is independent from the hormonal status of the animal, except for nAChR α7, which is less present during pregnancy. The epithelium expresses M1, M3, nAChR α7 (data from laser-assisted microdissection) and nAChR α3 (ultrastructural investigation of reporter mice). MR dominate over nAChR in increasing [Ca(2+)](i) with being M3 the major, but not sole subtype driving this effect. The general nAChR inhibitor mecamylamine enhances muscarinic and purinergic responses. SIGNIFICANCE: In conclusion, the murine oviduct is endowed with a multiplicity of muscarinic and nicotinic receptors subtypes that, with respect to regulation of [Ca(2+)](i), are inversely linked to each other. The major, but not sole, cholinergic receptor driving increase in [Ca(2+)](i) is M3.
Assuntos
Cálcio/metabolismo , Tubas Uterinas/metabolismo , Receptores Colinérgicos/análise , Receptores Colinérgicos/genética , Animais , Tubas Uterinas/ultraestrutura , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Receptores Colinérgicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
In the oviduct, the integrity of oocyte and sperm transport, fertilization, and early embryonic ontogenesis is essential for successful reproduction. Up to now, most of the knowledge on oocyte and sperm transport, gamete interaction and embryonic development has in most cases been gained exclusively by in vitro studies. In addition, especially the mechanisms of gameto-maternal interaction and embryo-maternal communication in the oviduct are still unknown. Recent techniques of live cell imaging and digital videomicroscopy allow for the first time to provide actual new insights in the mechanisms of sperm transport, sperm storage, oocyte transport, fertilization, gameto-maternal interaction and embryo-maternal crosstalk under near in vivo conditions. Detailed knowledge of these important events in the oviduct is the prerequisite to develop new therapeutic concepts for subfertility and infertility and to increase the success rates of the actual techniques of assisted reproduction (ART). Additionally the effects of drugs and hormones used in ART can be effectively studied using a functional oviductal epithelium. The guidelines for live cell imaging in the oviduct presented here should enable researches to establish a functional digital analysis system which allows to study physiological and pathological events in the oviduct under near in vivo conditions.
Assuntos
Microscopia de Vídeo/métodos , Oviductos/citologia , Animais , Bovinos , Sobrevivência Celular , Desenho de Equipamento , Feminino , Camundongos , Microscopia de Vídeo/instrumentação , Oviductos/ultraestruturaRESUMO
The transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. Because nonneuronally produced acetylcholine influences ciliary beating in the trachea via the muscarinic receptors M2 and M3, we supposed that components of the cholinergic system may also modulate ciliary activity in the oviduct. To address this issue, we analyzed the expression profile of muscarinic receptors (CHRMs) in the murine oviduct by RT-PCR and assessed ciliary beat frequency (CBF) and cilia-driven particle transport speed (PTS) on the mucosal surface of opened oviductal segments in correlation with histomorphological investigations. RT-PCR of laser-assisted microdissected epithelium revealed expression of Chrm subtypes Chrm1 and Chrm3. In opened isthmic segments, particle transport was barely seen, correlating with a significantly lower number of ciliated cells compared to the ampulla. In the ampulla, basal PTS and CBF were high (71 µm/sec and 21 Hz, respectively) both in cycling and pregnant wild-type mice and in mice with targeted deletion of the Chrm genes Chrm1, Chrm3, Chrm4, and Chrm5. In contrast to the trachea, where basal ciliary activity was low and largely enhanced by muscarinic stimulation, muscarinic agonists and antagonists did not affect the high ampullar PTS. Our results imply that this high oviductal autonomous ciliary activity is independent from the intrinsic cholinergic system and serves to maintain optimal clearance of the tube throughout all stages of the estrous cycle and early pregnancy.
Assuntos
Ciclo Estral/metabolismo , Tubas Uterinas/citologia , Tubas Uterinas/metabolismo , Gravidez/metabolismo , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Animais , Cílios/metabolismo , Feminino , Camundongos , Camundongos Knockout , Agonistas Muscarínicos , Antagonistas MuscarínicosRESUMO
Oocyte maturation is a prerequisite for successful fertilization. Growing evidence suggests that not only the oocyte but also the surrounding zona pellucida has to undergo maturational changes. In the pig, two-dimensional electrophoretic analysis demonstrated an acidic shift of the zona pellucida glycoproteins of about 1.5-2.0 pH units during the maturation process. These findings were corroborated by histological studies that indicated the synthesis of acidic glycoconjugates in the cumulus cells and an increased occurrence of acidic glycans in the zona pellucida after oocyte maturation. In order to provide structural data on prepuberal zona pellucida N-glycosylation, N-glycans were released from prepuberal zona pellucida glycoproteins by N-glycosidase F and studied by mass spectrometry before and after desialylation and treatment with endo-beta-galactosidase. Our results verified the presence of high-mannose-type Man(5)GlcNAc(2) compounds as well as diantennary N-glycans as major neutral species, whereas sialylated diantennary and triantennary species constituted the dominant non-sulfated acidic sugar chains. The major acidic N-glycans of prepuberal animals, however, represented mono-sulfated diantennary, triantennary and tetraantennary oligosaccharides carrying, in part, N-acetyllactosamine repeating units as well as additional Neu5Ac or Neu5Gc residues. Glycans comprising more than one sulfate residue were not detected. In contrast to the literature data on zona pellucida glycoprotein-N-glycans of cyclic animals, our data thus reveal a lower degree in glycan sulfation of the prepuberal zona pellucida.
Assuntos
Polissacarídeos/química , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Zona Pelúcida/metabolismo , Animais , Sequência de Carboidratos , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Suínos , Glicoproteínas da Zona PelúcidaRESUMO
Using a digital videomicroscopic analysis system in the bovine, we showed that the mechanisms of transport caused by ciliary beating are distinctly different in ampulla and isthmus of the oviduct. The average particle transport speed (PTS) in the oviduct (mean, 133 microm/sec) does not differ in the cycle (metestrus) and during pregnancy after implantation, but it is locally modulated at the site of the embryo. Using videomicroscopy, we were able to document that after entering the ampulla, the cumulus-oocyte complex (COC) is not transported by ciliary beating down the oviduct, but firmly attaches to the ampullar epithelium. This attachment is mediated by the cumulus cells. However, when a COC is degenerated, it is floating in the oviductal lumen. As soon as a vital COC is in the ampulla, the sperm bound in the sperm reservoir of the ampullar isthmic junction leave the reservoir and hurry to the oocyte. When a sperm has penetrated the zona pellucida, the COC detaches and continues its migration. Quantitative measurements showed that the early embryo is able to locally downregulate PTS during its migration down the oviduct. It locally changes the pattern of vascularization and induces the formation of secretory cells. Our studies imply that the oviductal epithelium is able to select vital oocytes. The early embryo is able to induce the formation of secretory cells, modify vascularization, and downregulate speed of transport, thus creating the prerequisite for the first embryo-maternal communication in the oviduct.