Caprine Ovary Research Articles

Effects of differently incubated cupric oxide nanoparticles on the granulosa cells of caprine ovary in vitro

In the nanoscience metal and metal oxide, nanoparticles have a prominent place because of their vast applications. Recent finding shows that in addition to size, there are other critical factors governing the biological response of nanoparticles. These factors include surface chemistry and shape that influences solubility, rate of diffusion, drug delivery, melting temperature, and colour of the nanoparticles. It is thus the present study that was aimed to investigate the effect of temperature on the shape and size of nanoparticles and related cytotoxicity of these particles on ovarian granulosa cells. Cupric oxide nanoparticles (CuONPs) were synthesized using a simple, efficient, and reproducible precipitation method involving the reduction of Cu metal salt with sodium hydroxide and then incubation of the precipitates at 70°C for 5h. Subsequently, this prepared sample was divided into 3 subsamples and incubated at 3 different temperatures, i.e. 70°C, 150°C, and 350°C for 5h to study the effect of temperature on the particles. The products were characterized by XRD, FTIR, HRTEM, and FESEM. Characterization of the particles revealed that all particles were monoclinic crystalline in nature and had a size range from 9 to 60nm. Particles were of different shapes: spherical, needle, and capsule. The toxicity of each particle was determined on granulosa cells by exposing cells for 24h at 2 different doses. Toxicological results showed the size and shape-related toxicity of nanoparticles where spherical shapes were significantly more toxic than capsule-shaped particles.

Chi-miR-4110 promotes granulosa cell apoptosis by targeting Sma- and Mad-related protein 2 (Smad2) in the caprine ovary.

Follicular atresia mainly results from the apoptosis of granulosa cells (GCs). Whilst our previous investigations examined the role of chi-miR-4110 in regulating ovarian function, the present study detected the role of chi-miR-4110 in GC development. We transfected caprine GCs cultured in vitro with chi-miR-4110 mimics. Results revealed that chi-miR-4110 decreased mRNA and protein levels of Smad2 by targeting its 3′-untranslated region (3′UTR). FoxC1 and Sp1 mRNA and protein levels markedly increased, whereas those of bHLHe22 significantly decreased (P<0.01 or 0.05) in GCs transfected with the chi-miR-4110 mimics. Further studies revealed a significantly higher number of apoptotic cells in GCs transfected with the chi-miR-4110 mimics (P< 0.05) than in GCs transfected with mimics negative control. GCs transfected with the chi-miR-4110 mimics exhibited significantly increased mRNA and protein levels of the pro-apoptotic gene Bax (P<0.01) and significantly decreased expression levels of the anti-apoptotic gene BCL-2 (P<0.01). Smad2 interference (Si-1282) results were consistent with those of the chi-miR-4110 mimics. Previous reports and our results showed that chi-miR-4110 increases Sp1 expression by repressing Smad2. The increase in Sp1 induces p53-upregulated modulator of apoptosis, which increases the relative abundance of Bax and causes caprine GC apoptosis. Our findings may provide relevant data for the investigation of miRNA-mediated regulation of ovarian functions.

N-acetyl cysteine-mediated effective attenuation of methoxychlor-induced granulosa cell apoptosis by counteracting reactive oxygen species generation in caprine ovary.

Methoxychlor (MXC), an organochloride insecticide, is a potent toxicant-targeting female reproductive system and known to cause follicular atresia by inducing apoptosis within granulosa cells. Oxidative stress plays a pivotal role in apoptosis; thus, this study focuses on the ameliorative action of N-acetyl cysteine (NAC) on MXC-induced oxidative stress and apoptosis within granulosa cell of caprine ovary. Classic histology, fluorescence assay, and biochemical parameters were employed to evaluate the effect of varied concentration of NAC (1, 5, and 10 mM) on granulosa cell apoptosis after 24, 48, and 72 h exposure duration. Histomorphological studies revealed that NAC diminished the incidence of apoptotic attributes like condensed or marginated chromatin, pyknosis, crescent-shaped nucleus, empty cell spaces, and degenerated cellular structure along with the presence of cytoplasmic processes within granulosa cells in dose- and time-dependent manner. NAC significantly downregulated the percentage of MXC-induced granulosa cell apoptosis within healthy ovarian follicle with its increasing dose, maximum at 10 mM concentration. It also significantly (p < 0.05) upregulated the activity of antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, along with ferric reducing antioxidant power further declining lipid peroxidation in the MXC-treated caprine ovary. The results revealed a negative correlation between apoptosis frequency and antioxidant enzymes' activity (rCAT = -0.67, rSOD = -0.56, rGST = -0.31; p < 0.05) while a positive correlation was observed with lipid peroxidation (r = 0.63; p < 0.05) after NAC supplementation. Thus, NAC supplementation reduces the MXC-generated oxidative stress that perhaps declines the ROS generating signal transduction pathway of apoptosis, thereby preventing MXC-induced granulosa cell apoptosis and follicular atresia. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 156-166, 2017.

Expression of angiotensin II receptors in the caprine ovary and improvement of follicular viability in vitro.

This study aimed to evaluate mRNA levels of angiotensin II (ANG II) receptors (AGTR1 and AGTR2) in caprine follicles and to investigate the influence of ANG II on the viability and in vitro growth of preantral follicles. Real-time polymerase chain reaction (PCR) was used to quantify AGTR1 and AGTR2 mRNA levels in the different follicular stages. For culture, caprine ovaries were collected, cut into 13 fragments and then either directly fixed for histological and ultrastructural analysis (fresh control) or placed in culture for 1 or 7 days in α-minumum essential medium plus (α-MEM+) with 0, 1, 5, 10, 50 or 100 ng/ml ANG II. Then, the fragments were destined to morphological, viability and ultrastructural analysis. The results showed that primordial follicles had higher levels of AGTR1 and AGTR2 mRNA than secondary follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA than their respective cumulus-oocyte complex (COCs). After 7 days of culture, ANG II (10 or 50 ng/ml) maintained the percentages of normal follicles compared with α-MEM+. Fluorescence and ultrastructural microscopy confirmed follicular integrity in ANG II (10 ng/ml). In conclusion, a high expression of AGTR1 and AGTR2 is observed in primordial follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA. Finally, 10 ng/ml ANG II maintained the viability of caprine preantral follicles after in vitro culture.

Cloning and expression of caprine KIT gene and associations of polymorphisms with litter size

The full coding region of KIT mRNA was cloned from the caprine ovary. The results showed the caprine KIT cDNA (GenBank accession number KF364483) contained a 2925-bp open reading frame encoding a protein with 974 amino acid residues. BLAST analysis revealed that the caprine KIT protein had high similarity with that of four species: Ovis aries (99%), Bos taurus (99%), Sus scrofa (94%) and Homo sapiens (90%). The KIT mRNA expression pattern showed that KIT mRNA was expressed highly in kidney, ovary, uterus and breast. Two single nucleotide polymorphisms (g.88430T &gt; A and g.120466G &gt; A) in the caprine KIT gene were detected by PCR–restriction fragment length polymorphism (RFLP) and DNA sequencing in 735 goats of Xinong Saanen, Guanzhong and Boer breeds. The g.88430T &gt; A mutation was a missense mutation (Tyr &gt; Asn at position 409 amino acid of KIT). The association study has been done by jointly analysing all data in one analysis. The result showed that individuals with TT and TA genotypes had their litter size increased by 0.11 and 0.09, respectively, compared with those with AA genotype at the g.88430T &gt; A locus for three goat breeds (P &lt; 0.05). Further analysis revealed that combined genotype TTAA was better than the others for litter size in three goat breeds. Therefore, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the KIT gene could serve as a genetic marker for litter size in goat breeding.

The expression and localization of leptin and its receptor in goat ovarian follicles

Leptin, a hormone that was originally identified in adipocytes, has been implicated in the regulation of ovarian folliculogenesis through endocrine, autocrine and/or paracrine mechanisms. The aim of this study was to investigate the expression patterns of leptin (LEP) and its receptor (LEPRb) in different types of ovarian follicular cells from goats. In small follicles, the expression levels of LEP were higher (P<0.001) in granulosa cells than in theca cells, cumulus cells and oocytes. The expression of LEP in granulosa cells was higher (P<0.001) in small follicles than in large follicles. In large follicles, the expression of LEPRb was higher (P<0.05) in granulosa cells than in theca cells, cumulus cells and oocytes. Higher expression (P<0.05) of LEPRb was detected in granulosa cells isolated from large follicles than in granulosa cells isolated from small follicles. Immunohistochemical analyses revealed the presence of the LEP and LEPR proteins in follicles at all stages of development. The most intense staining for LEP and LEPR was observed in the cytoplasm of oocytes and the surrounding granulosa cells. In conclusion, it was demonstrated that leptin and its receptor are expressed at both the mRNA and protein levels in goat ovarian follicles. Furthermore, the presence of a leptin signaling system in the caprine ovary suggests a potential regulatory role for leptin in follicular development and the maturation of goat oocytes.

Molecular cloning, tissue expression and SNP analysis in the goat nerve growth factor gene

In this study, we cloned the full coding region of NGF gene from the caprine ovary. Result showed the caprine NGF cDNA (GenBank Accession No. JQ308184) contained a 726bp open reading frame encoding a protein with 241 amino acid residues. Bioinformatic analysis indicated that caprine NGF amino acid sequence was 83-99% identical to that of mouse, pig, dog, human and bovine. It was predicted that caprine NGF contained nine serine phosphorylation loci, four threonine phosphorylation loci and nine specific PKC phosphorylation loci. The NGF mRNA expression pattern showed that NGF gene was expressed highly in ovary. This work provided an important experimental basis for further research on the function of NGF in goat. A single nucleotide polymorphism (A705G) in the coding region of NGF gene was detected by PCR-RFLP and DNA sequencing in 630 goats of three breeds. The frequencies of G allele were 0.52-0.61, and frequencies of A allele were 0.48-0.39 for SN, GZ and BG breeds, respectively. The does with GG genotype had higher litter size than those with GA and AA genotypes (P<0.05). Hence, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the NGF gene could serve as a genetic marker for litter size in goat breeding.

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.

Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol

The caprine ovary is a rich source of potentially viable immature oocytes enclosed in preantral follicles (PF). Previous experiments showed that these oocytes can be successfully cryopreserved in ovarian tissue of several species. However, until now, no information about the caprine PF cryopreservation is available in the literature. The aim of the present research was to evaluate the structural and ultrastructural characteristics of caprine PF after treatment and cryopreservation of ovarian tissue with 1.5 and 3 M dimethylsulphoxide (DMSO) and propanediol (PROH). One fragment of ovarian tissue was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four fragments were equilibrated at 20 °C/20 min in 1.8 ml of minimum essential medium (MEM) containing 1.5 or 3 M DMSO or PROH for the toxicity test, and the other four fragments were slowly frozen in each cryoprotectant at the concentrations previously described. After toxicity test and freezing/thawing procedures, the ovarian fragments were fixed for histological examination. The results showed that after toxicity test and cryopreservation of ovarian tissue using both cryoprotectants, the percentage of normal PF was less ( P<0.05) as compared with the control group. The present study revealed that the percentage of normal PF after toxicity test and cryopreservation in 1.5 M DSMO was significantly greater ( P<0.05) as compared with results obtained with 3 M DMSO or 1.5 and 3 M PROH. This result was confirmed by transmission electron microscopy, which showed that the PF were preserved in a higher quality state with 1.5 M DMSO. In conclusion, the present study demonstrated that caprine PF can be cryopreserved in ovarian tissue using 1.5 M DMSO.

Isolamento mecânico de folículos ovarianos pré-antrais em cabras

Com o objetivo de isolar mecanicamente folículos pré-antrais caprinos, 52 ovários de cabras sem raça definida (SRD) foram coletados em matadouros ou extraídos por ovariectomia. O protocolo utilizado para o isolamento dos folículos, denominado procedimento mecânico direto (PMD), consistiu no corte do tecido ovariano em pequenos fragmentos, utilizando-se um cortador de tecidos. Em seguida, os fragmentos obtidos foram dissociados mecanicamente com pipetas de pasteur (1600mim e 600mim de diâmetro) e a suspensão resultante, contendo os fragmentos ovarianos, foi sucessivamente filtrada em malhas de náilon (500mim e 100mim). A realização deste procedimento permitiu isolar um número médio (± EP) de 1.067 ± 160 folículos por ovário. Com a finalidade de aumentar o número de folículos pré-antrais isolados por ovário, foi utilizado um procedimento mecânico complementar (PMC), que consistiu em submeter os fragmentos retidos nas malhas de 500mim e 100mim às etapas descritas no PMD. A utilização desses fragmentos resultou em um incremento médio de 5,2% no total de folículos isolados por ovário. O número médio (± EP) de folículos isolados por ovário, utilizando-se o procedimento mecânico (PMD + PMC), foi 1.126 + 163. Independentemente do procedimento utilizado, observou-se uma grande variação individual no número de folículos isolados por ovário. Os limites mínimo e máximo desta variação foram 53 e 7.482 folículos, respectivamente. O tamanho dos folículos isolados variou de 15,0mim a 92,7mim. É viável a utilização do procedimento mecânico, descrito pela primeira vez, para o isolamento de folículos ovarianos pré-antrais caprinos.

Ultrastructure of thecal components in caprine ovary

The fine structure of the thecal components of normally developing preantral and antral follicles of goat ( Capra hircus) ovaries was studied. The theca externa was largely composed of connective tissue (fibroblasts and adipocytes). The fine structure of theca externa cells showed the presence of large vesicular mitochondria and a mesh of rough endoplasmic reticulum. A few lipid vacuoles/bodies were also observed. The cytoplasm of theca interna cells contained numerous profiles of smooth endoplasmic reticulum, vesicular mitochondria with tubular cristae and free ribosomes. The ultrastructural organization of caprine theca largely resembles that of other Bovidae species.

Histomorphological changes of the caprine ovary relative to the stages of the estrous cycle

Thirty Marwari goats (2 to 2.5 yr of age) were slaughtered at six different stages of the estrous cycle, five were sacrificed on the first day (D1), five each on the second day (D2), third day (D3), ninth day (D9) and fifteenth day (D15), and five goats which had smooth ovaries (SO). Histomorphological observations indicated that ovarian follicular numbers and diameter from ipsilateral and contralateral sides did not influence different stages of the estrous cycle. The total follicle population was significantly higher in the ipsilateral ovary. Width of thecal layer and tunica albuginea varied according to ovarian status and physiology. Cellular type, cellular activity and staining intensity in ovaries were changed according to the physiological condition during different stages of the estrous cycle. Ovarian activity of Marwari goats during estrous cycle stages were similar to other breeds of goats.

The in vitro and in vivo anti-retrovirus activity, and intracellular metabolism of 3′-azido-2′,3′-dideoxythymidine and 2′,3′-dideoxycytidine are highly dependent on the cell species

Cell lines derived from different species show striking differences in their sensitivity to the cytostatic and anti-retrovirus activity, as well as the intracellular metabolism, of 3'-azido-2',3'-dideoxythymidine (AzddThd) and2',3'-dideoxycytidine (ddCyd). AzddThd and ddCyd are considerably more cytostatic to human (i.e. Raji, Molt/4F, ATH8) cell lines than murine (i.e. L1210) cells. The intracellular levels of AzddThd 5'-triphosphate and ddCyd 5'-triphosphate formed do not seem related to the cytostatic effects achieved by these compounds. In human lymphoid (ATH8, Molt/4F) and caprine ovary (Tahr) cells AzddThd accumulates as its 5'-monophosphate (AzddTMP), whereas in murine leukemia (L1210) cells it is readily metabolized to the 5'-triphosphate (AzddTTP). The rapid conversion of AzddThd to AzddTTP in murine cells may explain why AzddThd has a pronounced activity against Moloney murine sarcoma virus (MSV)-induced transformation of murine C3H cells in vitro and MSV-induced tumor development in newborn NMRI mice in vivo. In contrast, ddCyd has not much activity in these murine assay systems, and this may seem related to the poor conversion of ddCyd to its 5'-triphosphate in murine cells. In human cells, however, ddCyd is more extensively phosphorylated to its 5'-triphosphate than in murine cells. When [ 3H]AzddThd and [ 3H]ddCyd were compared for their metabolism in ATH8 and Molt/4F cells, little [ 3H]AzddTTP was formed even after a 48-hr incubation period, whereas under the same conditions substantial levels of [ 3H]ddCTP built up gradually. Thus, much higher ddCTP than AzddTTP levels were achieved in human lymphoid cells, an observation that may be particularly relevant from a therapeutic viewpoint.