56 research outputs found
Chemical imaging of canine oviducts during the post-ovulatory period
No full textIt is well known that bitches present a peculiar oocyte maturation, characterized by ovulation of immature oocytes and a long period of viability in the oviduct. Thus, the oviduct in this species plays an essential role in oocyte maturation, in addition to fertilization and early stage embryo development. Different approaches have been used in order to identify the factors involved in each step (from resumption of meiosis through development of embryos) and recently a new technique was used for spatial identification of oviductal proteins in the domestic cat (1). With the purpose to contribute to the understanding of these factors, we applied MALDI imaging mass spectrometry (MALDI-IMS) to obtain protein profiling and imaging of canine oviducts. Reproductive tracts were collected from 4 bitches in estrus (cross-breed, 2 to 6 years old) undergoing routine ovariohysterectomy. Post-ovulatory period was confirmed by blood serum progesterone concentrations (P4 mean±SD: 15.5±2.5) and ovarian morphology. The oviducts were carefully dissected, divided into three segments (distal to the ovary-isthmus, proximal to the ovary-infundibulum and the mid-section between the two-ampulla; further confirmed by histology), snap-frozen in liquid nitrogen and stored at -80oC until use. Then, they were sectioned (11 μm) in a cryostat and fixed on ITO (indium tin oxide) conductive glass slides, while serial sections were collected on microscope slides for haematoxylin and eosin staining. For MALDI-IMS, samples were coated with a thin homogeneous layer of CHCA (α-Cyano-4-hydroxycinnamic acid) matrix using a nebulization device. MALDI images were acquired on an Autoflex III Smartbeam instrument (Bruker Daltonics) with 400 shots/spectrum, over the mass range of m/z 2 to 20 kDa in the positive ion mode. The spatial resolution of images was 80 μm. Mass spectra were characterized by abundant ions of m/z 2279, 2401, 3458 and 4976; which have been tentatively attributed to actin cytoplasmic 2, keratin type 1, neutrophil defensin 1 and thymosin β4, respectively. Actin has been detected in oviduct fluid of alpaca (2) and is related to epithelial cell renewal or secretory activity (3). As previously described in queens (1), defensin, keratin and thymosins are defense proteins that integrate the innate immune systems and are involved in the biological response to cellular damage. These data might contribute to piecing together the puzzle of factors that are involved in the peculiar aspects of the domestic dog reproductive physiology that might hamper in vitro embryo production.
1) Apparicio M, Santos VG, Rocha DFO et al. Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins. Reprod Domest Anim. 2017;52 (Suppl. 2):88–92.
(2) Apichela SA, Argañaraz ME, Zampini R et al. Biochemical composition and protein profile of alpaca (Vicugna pacus) oviductal fluid. Anim Reprod Sci. 2015,154:79-85
(3) Steffl M, Schweiger M, Sugiyama T et al. Review of apoptotic and non-apoptotic events in non-ciliated cells of the mammalian oviduct. Ann Anat. 2008,190: 46-52
Spermatozoa co-culture does not improve oocyte nuclear maturation rates in dogs
No full textIn canids, in order to overcome the limited efficiency of oocyte in vitro maturation1, several attempts mostly investigating different supplementations of the culture media, culture periods, and variation of the conditions over the course of the culture period (so-called bi-phasic media)2 have been made. In vivo, as canine oocytes are ovulated immature and remain an extended period within the oviducts, spermatozoa are present before full maturation occurs. Taken this into account, it was interesting to verify whether the presence of spermatozoa in in vitro culture medium could trigger meiosis resumption and improve maturation rates. For this purpose, ovaries were collected from bitches at different reproductive status (various breeds, age 1-7 years) by routine ovariohysterectomy and sliced in PBS-PVA to release the cumulus-oocyte complexes (COCs). Grade I COCs (n. 405) were obtained and randomly allocated in four systems with the base medium (BM) consisting of TCM-199 with antibiotics, 10% FBS, 2.2 mg/ml sodium bicarbonate and 20 l/ml pyruvic acid. Hormones were supplemented at the dose of 10 i.u/ml hCG (Sigma Chemical Co., MO, USA), 1μg/ml P4 (Sigma), 1μg/ml E2 (Sigma) as follows: in system A (n.110) oocytes were matured for 48 h in BM with hCG and for additional 24 h in BM with P4; in system B oocytes (n.118) were matured for 48 h in BM with hCG, P4 and E2 and for 24 h in BM with P4; in system A-S (n.92) and B-S (n.85) oocytes were cultured in system A and B, respectively, but with the addition of spermatozoa (5 x 106 sperm/ml) at 48 h of culture. COCs were incubated at 38C, 5% CO2 in air. At the end of maturation period (72 h), oocytes were denuded within 0.2% hyaluronidase solution (Sigma) by repeated pipetting and then, were stained with Hoechst 33342 (Sigma) for evaluation of meiotic configuration. Data were analyzed by ANOVA at p0.05. The percentage of degenerated oocytes significantly increased with the presence of spermatozoa (9%, 7.6%, 32.6%, and 28.3%, for systems A, B, A-S, and B-S, respectively; p<0.001). The overall sperm penetration occurred both in immature and mature oocytes, however a higher proportion of matured oocytes (60.5%, 23/38) were penetrated compared to the immature ones (22%, 4/18; p=0.0006). We can speculate that the potential influence of sperm penetration could be on cytoplasmic maturation, rather than nuclear maturation. This aspect should be further investigated and the fact that different periods of co-incubation might influence the results.
[1] Luvoni GC, Chigioni S, Allievi E, Macis D. Factors involved in vivo and in vitro maturation of canine oocytes. Theriogenology 2005; 63: 41–59.
[2] Apparicio M, Mostachio GQ, Motheo TF, et al. Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems. Reproduction, Fertility and Development 2015; 27:1082–1087
Viability of feline preantral follicles in vitro cultured with Insulin Growth Factor and Epidermal Growth Factor supplemented medium
No full textIn vitro culture of ovarian preantral follicles (PF) has emerged as a potential reproductive technology for the production of mature oocytes capable of fertilization [1]. Advances concerning the role of several growth factors (GF) on in vitro activation of primordial follicles have been described. The addition of EGF (Epidermal Growth Factor) and IGF (Insulin-like Growth Factor) in the in vitro culture of PF of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells (GC) [2]. However, there are only few reports regarding the use of these factors on feline PF in vitro culture. We previously demonstrated that IGF-1 exerts a positive influence on follicular development [3] and has been recently shown that EGF sustains in vitro primordial follicle viability in the prepubertal cat ovary [4]. Thus, the aim of this study was to investigate the effect of a combination of IGF and EGF on in vitro growth and GC viability of PF collected from domestic cats. A total of 64 PF characterized by multi-layer granulosa cells and theca cells with 150–200 μm of diameter were isolated and individually cultured for 6 days (T6) in minimum essential medium (MEM; Sigma-Aldrich Co., St. Louis, LO, USA) supplemented with IGF+EGF (100 ng/ml each; Sigma) or without GF (control). Data from follicular and oocyte diameters were submitted to Kolmogorov–Smirnov. Mean values of the increase in follicular size were analysed by Student–Newman–Keuls test, and GC viability was analysed by chi-square test (P 0.05; Sigma). The GC of PF cultured with GF maintained a greater viability (fluorescein diacetate staining; Sigma) than those of PF cultured without (84.3% and 68.7% respectively; P<0.05). These data suggest that the addiction of IGF and EGF to the culture medium promotes better conditions to the in vitro development of preantral follicles of cats. It remains to investigate the precise role of the single growth factor to establish optimal culture conditions.
[1] Demeestere I, Centner J, Gervy Y, et al. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction 2005; 130:147-56.
[2] Silva J R V, Van Den Hurk R, Figueiredo J R. Ovarian follicle development in vitro and oocyte competence: advances and challenges for farm animals. Domest Anim Endocrinol 2016, 55:123-5.
[3] Alves EA, Padilha L, Savi PA, et al. In vitro survival of follicles recovered from queens at different stages of estrous cycle and cultured with IGF-1. Reprod Domest Anim 2012; 47 (Suppl. 6):109-12.
[4] Fujihara M, Comizzoli P, Keefer CL, et al. Epidermal growth factor (EGF) sustains in vitro primordial follicle viability by enhancing stromal cell proliferation via MAPK and PI3K pathways in the prepubertal, but not adult, cat ovary. Biol Reprod 2014; 90:86
Canine and feline oocyte lipid profile by Matrix-Assisted Desorption Ionization time-of-flight Mass Spectrometry (MALDI-TOF MS)
No full textOBJECTIVES AND METHODS. Canine and Feline oocytes present an elevate cryosensibility, mostly due to their high lipid content (1). It is believed that the presence of intracellular lipid droplets could be responsible for uneven intracellular ice formation which could affect the freezing-thawing process (2). Studies on the lipid composition of oocytes from these species may contribute with the improvement of the cryopreservation process as well as to the advancement of in vitro culture conditions. Thus, the aim of this work is to apply the technique of matrix-assisted desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to obtain the lipid profiles of single canine and feline oocytes and compare with the lipid profiles obtained for the bovine oocytes, a species commonly used for cryopreservation studies. Ovaries were collected from three diestrous bitches (various breeds, age 1-7 y) and one queen by routine ovariohysterectomy and sliced in PBS-PVA to release the cumulus-oocyte complexes (COCs). Oocytes were graded according to morphological characteristics and only grade I COCs were selected and stored in PBS solution at -20C until analysis. For MALDI-TOF MS analysis, samples were washed in H2O/MeOH 1:1 (v/v) and placed (1 to 3 oocytes/spot) in the MALDI target plate. Samples were then covered with 2.5 dihydroxybenzoic acid (DHB) as the organic matrix. A Synapt HDMS (Waters Corp. Milford, MA, USA) equipped with a MALDI source was used for the experiments. All mass spectra were manually collected for around 1 min in the positive ion mode, at the m/z range of 700-1000. Mass spectra collected from individual embryos were processed using the software MassLynx 4.1 (Waters Corp. Milford, MA, USA).
RESULTS. This is the first report about lipid fingerprint of canine and feline oocytes by MALDI-TOF MS. The technique has been already reported for bovine oocytes and embryos, as well as human and sheep oocytes (3). Besides the analysis workflow simplicity (no lipid extraction), intact complex lipid species such as sphingomyelins (SM), phosphocholines (PC) and triacylgycerols (TAG) can be detected because lipids are easily ionizable by MALDI using DHB as the matrix. By gas chromatography (GC), traditionally used for lipid analysis in gametes and embryos, only fatty acyl residues can be detected and structural information of lipids is lost. Mass spectra of bitch and queen oocytes were similar and characterized mainly by intense ions correspondent to the m/z values of PC containing 34 carbons (of m/z 780.7 to 786.7) and 36 carbons (of m/z 808.7 to 812.7), but mainly PC (34:1) of m/z 782.8 (sodiated PC 34:1), m/z 723.7 (fragment of PC 34:1) and 760.8 (protonated PC 34:1), which is the main phospholipid present in the cellular membrane and this fact has been also observed for bovine oocytes (3). Regarding the TAG species, m/z values correspondent to species containing 52 and 54 carbons were more prominent in canine and especially in feline oocytes, compared to the bovine (3).
CONCLUSIONS. Lipid attributions described herein must be confirmed by MS/MS experiments, but our data indicate that MALDI-TOF MS is able to detect the presence and the chemical structure (number of carbons present in the fatty acyl residues and unsaturations) of PC and TAG species in canine and feline oocytes. We envisage MALDI-TOF MS will become a relevant tool in studies aiming at improving canine and feline oocyte cryopreservation success.
(1) -Luvoni GC, Pellizzari P, Battocchio M. Effects of slow and ultrarapid freezing on morphology and resumption of meiosis in immature cat oocytes. J Reprod Fertil Suppl 1997; 51: 93-98.
(2) Luvoni GC. Gamete cryopreservation in the domestic cat. Theriogenology 2006; 66: 101-11.
(3) Ferreira CR, Saraiva SH, Catharino RR, Garcia JS, Gozzo FC , Sanvido GB, Santos LFA, Lo Turco EG, Pontes JHF, Basso AC, Bertolla RP, Sartori R, Guardieiro MM , Perecin F , Meirelles FV , Sangalli JR, Eberlin MN. Single embryo and oocyte lipid fingerprinting by mass spectrometry. Journal of lipid research 2010; 51: 1218-1227
MALDI imaging mass spectrometry (MALDI-IMS): a new approach for spatial identification of proteins in feline oviducts
No full textThe knowledge and understanding of many reproductive processes have improved significantly over the years, especially after the development of tools for protein identification1. The traditional approach for spatial identification of proteins requires a prior knowledge of the target entities and narrows the number of targets that can be monitored simultaneously1. For that reason, MALDI imaging mass spectrometry (MALDI-IMS) once it is able to directly scan the target tissue, is considered an outstanding approach for molecular profiling and imaging. With the purpose to identify the factors involved in early stages of embryo development in the domestic cat, this technology was used for the first time to analyse the spatial distribution of proteins in oviducts of queens. Reproductive tracts were collected from 2 queens (cross-breed, 2 and 4 years old) undergoing routine ovariohysterectomy. The oviducts were carefully dissected, divided into three segments (distal to the ovary, proximal to the ovary and the mid-section between the two), snap-frozen in liquid nitrogen and stored at -80oC until use. Then, they were sectioned (11 μm) in a cryostat and fixed on ITO (indium tin oxide) conductive glass slides while serial sections were collected on microscope slides for haematoxylin and eosin staining. For MALDI-IMS, samples were coated with a thin homogeneous layer of CHCA (α-Cyano-4-hydroxycinnamic acid) matrix using a nebulization device. MALDI images were acquired on an Autoflex III Smartbeam instrument (Bruker Daltonics) with 400 shots/spectrum, over the mass range of m/z 2 to 20 kDa in the positive ion mode. The spatial resolution of images was 80 μm. The molecular images obtained in this study show that protein distribution was different in the oviductal infundibulum, ampulla, and isthmus identified by histology. Mass spectra was characterized by abundant ions of m/z 1259, 4939, 4960 and 10626. Based on the literature2 these ions might correspond to keratin, thymosin β10, thymosin β4 and S100, respectively. S100 proteins are calcium modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. Keratin and thymosins are involved in the biological response to tissue damage. In women, this defence system is adapted to operate in the genital tract, being altered according to the hormonal and cellular changes that occur during menstruation (characterized by cell death and necrosis)3. Protein attributions described herein must be confirmed by MS/MS experiments, but our results indicate that it is possible to use this technology to elucidate key molecular processes involved in the reproductive physiology of carnivore species.
[1] Lagarreigue M, Lavigne R, Guevel B, et al. Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry: A Promising Technique for Reproductive Research. Biology of Reproduction 2012; 86(3): 1–11. [2] McDonnell LA, Walch A. Stoeckli M, Corthals GL. MSiMass list: a public database of identifications for protein MALDI MS imaging. Journal of proteome research 2013, 13 (2), 1138-1142. [3] Horne AW, Stock SJ, King AE. Innate immunity and disorders of the female reproductive tract. Reproduction 2008; 135(6): 739-749
In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1
No full textContents Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 +/- 22.1 mu m, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 +/- 14.4 mu m, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 +/- 15.9 mu m, 63.6%, respectively) than that cultured without IGF-1 (26.7 +/- 14.4 mu m, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Estadual Paulista, Dept Med Vet Prevent & Reprod Anim, Setor Reprod & Obstet Vet, FCAV,UNESP, BR-14884900 Jaboticabal, SP, BrazilUniv Milan, Dipartimento Sci Vet Salute Prod Anim & Sicurezza, Milan, ItalyUniv Estadual Paulista, Dept Med Vet Prevent & Reprod Anim, Setor Reprod & Obstet Vet, FCAV,UNESP, BR-14884900 Jaboticabal, SP, Brazi
In vitro survival of preantral follicles recovered from queens at different stages of estrous cycle and cultured with IGF-1
No full textOBJECTIVE AND METHODS. In vitro growth of preantral follicles may supply high numbers of competent oocytes that can be destined to in vitro embryo production. However, optimal culture systems that support follicular and oocyte development in vitro have not yet been defined. Ovarian follicular development is regulated mainly by endocrine, autocrine, and paracrine factors and follicles are exposed to specific hormonal environments during the different stages of the estrous cycle. The intraovarian insulin-like growth factor-I (IGF-1) regulates in vivo follicular development and there are important evidences that it stimulates in vitro growth of preantral follicles in domestic animals (1). Feline preantral follicles have been previously cultured in vitro (for review 2) and it has been suggested that IGF-1 enhances oocyte metabolism (3). However, no information are available on the response of preantral follicles recovered in different phases of the estrous cycle to cultural conditions. Thus, the aim of this study was to investigate the in vitro survival of preantral follicles recovered from ovaries of queens in follicular or luteal phase of the estrous cycle and cultured in presence of IGF-1.
Twelve queens were housed with 12 hours of light and submitted to estrous induction with IM injection of 100 UI eCG (Novormon®- Intervet) and 100 UI hCG (Vetecor®- Hertape Calier) 82 hours later (4). Six queens were spayed 96 h after eCG administration (follicular phase), the others 36 h after the hCG injection (luteal phase). Estrous phases were confirmed by vaginal cytology prior to the surgical procedure and macroscopic evaluation of ovarian structures after excision.
Preantral follicles surrounded by complete basal membrane and containing more than one layer of granulosa cells were retrieved from excised ovaries and selected as previously described (5). A total of 72 follicles were cultured for 6 days at 38.5 ̊C and 5% CO2 in air in Minimum Essential Medium (Sigma Chemical Co., USA) with IGF-1 100 ng/ml (Sigma) or without (control). Before and after culture, follicular diameter was recorded and follicular viability was assessed by fluorescein diacetate (FDA, Sigma) staining. Increase (%) in diameter was analyzed by Tukey’s and Fisher’s test, viability rates by Chi-square test (P<0.05).
RESULTS. After 6 days of culture, preantral follicles retrieved during follicular phase showed a significant increase in the size and a higher viability rate than those retrieved in the luteal phase of the estrous cycle (18.8% vs.11.5%; P= 0.0001 and 75% vs. 62.5%; P=0.004). However, when IGF-1 was added to the culture medium, follicles retrieved in follicular or luteal phase showed similar increase in diameter (14.9% vs.13.4%; P>0.05) and viability (73.8% vs. 76%; P>0.05). Regardless of the stage of the estrous cycle, overall results showed that the increase in diameter was not different in follicles cultured with or without IGF-1 (14.6% vs. 15.8%; P>0.05), but follicular survival was enhanced when IGF-1 was added to the culture medium (75% vs. 69.4%; P=0.0001).
CONCLUSIONS: Present data suggest that in vitro survival of preantral follicles is affected by the estrous stage of the donor and IGF-1 improves survival of follicles retrieved in luteal phase of the estrous cycle. Thus, the hormonal environment of the follicles within the ovary might impact their potential development when isolated and cultured. Further investigations on growth factors are needed to evaluate their effect on follicles with reduced in vitro developmental capability.
REFERENCES
(1) Giudice LC. Insulin-like growth factors and ovarian development. Endocr Rev 1992; 13: 641-669.
(2) Jewgenow K, Paris MCJ. Preservation of female germ cells from ovaries of cat species. Theriogenology 2006; 66: 93-100.
(3) Jewgenow K. Impact of peptide growth factors on the culture of small preantral follicles of domestic cats. Theriogenology 1996; 45: 889-895.
(4) Villaverde AI, Melo CM, Martin I, Ferreira TH, Papa FO, Taconeli CA, Lopes MD. Comparison of efficiency between two artificial insemination methods using frozen-thawed semen in domestic cat (Felis catus): artificial insemination in domestic cats. Anim Reprod Sci 2009; 114: 434-442.
(5) Lima AK, Bezerra MB, Oliveira LC, Figueiredo JR, Silva LDM. Isolamento e caracterização de folículos ovarianos pré-antrais em gatas domésticas (Felis catus). Rev Bras Reprod Anim 2003; 27: 396-397
Effect of Epidermal Growth Factor on in-vitro culture of feline preantral follicles
No full textIn vitro culture of preantral follicles would result in follicular development and production of a high number of competent
oocytes that can be destined to assisted reproduction programs. However, in vitro systems supporting complete follicular growth from early primordial stages have only been achieved in mice (I). The improvement of culture system is aimed at defining cultural conditions similar to the in vivo environment. Growth factors may act as autocrine or paracrine regulators to locally modulate follicular development. It has been shown that Epidermal Growth Factor (EGF) is a vigorous mitogen of follicular cells and induces follicular development (2). The aim of this study was to investigate
the effect of EGF on the in vitro development of preantral follicles recovered from domestic cat ovaries. Secondary follicles surrounded by complete basal membrane and containing more than one layer of granulosa cells were selected as previously described (3) and cultured for 6 days at 37°C and 5% C02 in air in Minirnum Essential Medium (Sigma Chemical Co., USA) with EGF 100 ng/ml or without (Control).
In vitro development was assessed by measuring the diameter of follicles and oocytes before and after culture. A significant increase in the size of follicles was observed when EGF was supplemented to the culture medium (increase
of 15.8%±2.53% vs 7.6%±5.2%; P<0.05), whether
oocyte diameter was not affected by cultural conditions. Viability of follicles was assessed at D6 by trypan blue staining and the results showed that follicle viability was also enhanced by EGF (75% vs 50%; P<O.05). In conclusion, feline preantral follicles respond to culture conditions and
these results prompt to extend the culture time in order to achieve the antrum formation. References 1) Demeestere et
al., Hum Reprod. 2002; 17:2152-9. 2) Morbeck et al., J Reprod Fertil. 1993;99:577-84.3) Lima et al., Rev Bras Reprod Anim 2003;27:396-7
Viability and growth of feline preantral follicles in vitro cultured with insulin growth factor and epidermal growth factor supplemented medium
No full textIn vitro culture of ovarian preantral follicles has emerged as a reproductive technology aimed at obtaining large amount of oocytes for in vitro embryo production. The addition of growth factors (GF) in the in vitro culture of preantral follicles of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells. However, there are only few reports regarding the use of these factors on feline preantral follicle in vitro culture. Thus, the aim of this study was to investigate the effect of a combination of IGF-1 and EGF on in vitro viability and growth of preantral follicles and enclosed oocytes collected from domestic cats. A total of 64 follicles characterized by multilayer granulosa cells were isolated and individually cultured for 6 days (T6) in minimum essential medium supplemented with IGF-1+ EGF (100 ng/ml each) or without (control). A higher percentage of follicles were viable after culture with GF than without, and an increase in size when IGF-1+ EGF were added to the medium (170 ± 32.4 μm (T0) vs. 201 ± 22.3 μm (T6); p .05). These data suggest that the addition of IGF-1 and EGF to the culture medium promotes the in vitro development of preantral follicles of cats
Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins
No full textWith the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used for the first time to describe the spatial localization of proteins in the oviducts of queens. Oviducts were obtained from two 2 and 4 years old cross-bred queens, divided into three segments, snap-frozen in liquid nitrogen and then stored at -80°C until use. Next, they were sectioned in a cryostat, fixed on ITO (indium tin oxide) conductive glass slides for MALDI-IMS and serial sections were collected on microscope slides for histology. As confirmed by histology, MALDI-IMS was able to show contrasting protein distributions in the oviductal infundibulum, ampulla and isthmus. Mass spectra were characterized by abundant ions of m/z 1,259, 4,939, 4,960 and 10,626, which have been tentatively attributed to keratin, thymosin β10, thymosin β4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calcium-modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. These results suggest that protein composition differs between segments of the cat oviduct, which corresponds to morphological changes within these sections. Further functional studies could elucidate the effects of these proteins on feline reproductive physiology
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