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Fine structures of the oocyte in relation to serum, follicular fluid steroid hormones and IGF-I in the ovulatory-sized follicles in one-humped camel camelus dromedarius.

Abstract

Bachground: The following study was carried out to determine the ultrastructural features of the oocyte of the ovulatory-sized follicles in relation to concentrations of steroids and IGF-I in the Follicular Fluid (FF) and serum in the dromedary camel.

Methods: Camel follicles with a clear and healthy appearance were categorized into three classes: follicles 10 to 13.9, 14-17.9 and 18-30 mm diameter. The FF and serum samples were assayed for estradio1-171I, progesterone and IGF-I. Recovered Cumulus-Oocyte Complexes (COCs) were prepared for transmission electron microscopy.

Results: The mean (-[+ or -]-5D) FF concentrations of progesterone and IGF-1 was significantly (p<0.05) higher in follicles 18 to 30 mm diameter compared to other groups of follicles. There was no difference in the mean ([+ or -]SD) serum estradio1-171, progesterone and IGF-I concentrations between camels with different ovulatory-sized follicles (p>0.05). Oocytes from follicles 18 to 30 mm diameter (group 3) showed more advanced signs of maturation including the disappearance of the nuclear envelope, increased number of microvilli in erect position, the increase in number and size of vesicles and more even distribution of the mitochondria throughout the ooplasm.

Conclusion: The final stages of oocyte maturation in dromedary camel is associated with increasing progesterone and IGF-I concentrations and constant high estradiol concentration in the follicular fluid which are paralleled with well-defined ultrastructural changes in oocytes.

Avicenna I Med Biotech 2014; 6(1): 57-61

Keywords: Camel, IGF-I, Oocyte, Steroids, Ultrastructure

Introduction

The camel, a popular and economically important species performs well under arid and semi-arid climate conditions, still remains one of the most neglected species in the field of scientific research. The reproductive efficiency of camels under natural conditions is being reported to be low (1). Various attempts have been made to improve the reproductive efficiency and genetic potential of dromedary camel (2). Techniques such as in vitro production (3) and cloning (4) in the dromedary camel have been successfully carried out with a low success rate. Therefore, there is a serious need to perform more basic research regarding the biology of follicular development and oocyte maturation in vivo. In addition, knowing the endocrinology and morphological aspects of the final stage of preovulatory follicle and oocyte maturation are the key elements for designing optimum in vitro conditions to develop oocyte and embryo culture systems.

Our study is the first to show the relationship between ultrastructure of the oocyte to hormone concentrations in the FF in Cainelus dromedarius (C. dromedarius). The ultrastructural changes of the immature oocytes (5) and oocytes from different follicle sizes (6) were described in the dromedary caml. Kafi et al (7) also described the ultrastructural features of the oocytes during in vitro maturation in the dromedary camel. Therefore, the following study was carried out to determine the ultrastructural features of the oocyte of the ovulatory-sized follicles associated with the concentrations of steroids and IGF-I in the FF and serum in the dromedary camel.

Materials and Methods

Ovaries were recovered from sexually mature non-pregnant camels (C. dromedarius) (6). During slaughtering, jugular blood samples were collected, centrifuged at 1400 for 30 min and stored in -20[degrees]C until analysis.

The ovulatory-sized follicles with a healthy and clear appearance were divided into three classes: 1) follicles 10-13.9 mm diameter, 2) follicles 1417.9 mm diameter and 3) follicles 18-30 mm diameter. The follicles were aspirated and the cumulus-oocyte complexes were isolated under stereomicroscope. COCs were prepared for transmission electron microscopy as previously described 6. The FF samples were centrifuged at 3000xg for 15 min, decanted and stored in small aliquots at -20[degrees]C for hormone assay.

Estradiol 17-[beta], progesterone and IGF-1 concentrations in the FF and serum were determined using a validated commercial radioimmunoassay kit (Immunotech kit, France). The differences in mean ([+ or -]SD) hormone concentrations were statistically compared using a one-way ANOVA. Duncan's multiple range test was applied to find the significant differences in multiple mean comparisons using the SPSS software. Correlations between serum and FF hormone concentrations were assessed by correlation analysis. The level of significance was set at p<0.05.

Results

The class size and the mean ([+ or -]SD) follicular fluid concentrations of estradiol- 17[beta], progesterone and IGF-I in different size follicles and the serum values of the hormones assayed are shown in table I. The mean ([+ or -]SD) follicular fluid concentrations of estradiol-1 713, progesterone and IGF-I were significantly different (p<0.05) in follicles 10-13.9 mm diameter than follicles with 18-30 mm diameter. Further, the mean ([+ or -]SD) FF concentrations of progesterone and IGF-I were significantly different (p<0.05) in follicles 14-17.9 mm diameter than that of follicles 18-30 mm diameter. There was no significant difference in the mean ([+ or -]SD) follicular fluid concentrations of estradio1-1713 between the follicles 14-17.9 and 18-30 mm diameter.

The Estrogen:Progesterone (E/P) ratio in the follicular fluid was significantly higher in follicles 10-13.9 mm diameter (59.07[+ or -]14.12) than in the FF in the follicles 14-17.9 mm diameter (18.90[+ or -] 5.19) and 18-30 mm diameter (4.07[+ or -]1.34) compared with 10 to 13.9 mm diameter. E/P ratio was not significantly different between follicles 1417.9 mm diameter and follicles 18-30 mm diameter (Table 1).

The concentrations of estradiol- I 713, progesterone and IGF-I in the serum were not correlated to those of the FF (r=0.23 for estradiol- I 713, r=-0.03 for progesterone and r=0.33 for IGF-1; p>0.05), respectively. Also there were no differences between serum concentrations of estradiol-1713, progesterone and IGF-1. of camels with different ovulatory-sized follicles (p>0.05).

Oocyte ultrastructure evaluation

Group 1-follicles 10-13.9 mm: The oocytes were surrounded by relatively expanded granulosa cells. Zona pellucida was homogenous containing nuprojections of cumulus cells. The nucleus was located eccentrically in half of the oocytes. The Perivitelline Space (PVS) was small with limited numbers of microvilli. Large numbers of different size vesicles were distributed through the oo-plasm. Clusters of mitochondria and Golgi complex were observed with peripheral distribution (Figure 1). Few numbers of lipid droplets were observed. Vesicles, mitochondria and lipid droplets were intermixed and located close to both vesicular and tubular endoplasmic reticulum in peripheral area.

Group 2-follicles 14-17.9 mm

Most ultrastructural features, in general, were similar to last group except that the PVS was slightly enlarged, the numbers of microvilli increased and the nucleus was peripherally located in all the oocytes (Figure 2).

Group 3-follicles 18-30 mm

All the oocytes showed no granulosa cells around. The number of microvilli was progressively increased (Figure 3). Cortical granules were also noted in the close proximity to the oolemma. The mitochondria were distributed through the ooplasm and the number and size of vesicles were observed to increase and distributed generally (Figure 4). The nuclear envelope was no longer apparent. The presence of the first polar body in the PVS was noted in one of the oocytes.

Discussion

The results of the present study showed that as the diameter of the ovulatory-sized follicle increases, the mean concentrations of estradio1-17[beta], progesterone and IGF-I in the FF concomitantly increases in the female dromedary camel. Similarly, Rahman (8) reported that concentrations of estra-diol-17[beta] and progesterone in the follicular fluid increased with the growth of the follicles in the female dromedary camel. In the equine, Spicer et al 9 showed that estradio1-17[beta] and IGF-I concentrations in the FF was greater in large ovulatory than in medium size follicles. In cattle, IGF-I stimulates estradiol synthesis which is important for final maturation of the preovulatory follicle and the oocyte (10). The concomitant increase of FF IGF-I and estradiol-17[beta], simultaneously with the increase in ovulatory-sized follicles in the present study suggests the presence of a similar relationship between IGF-I and estradiol-17[beta] in the female dromedary camel as it occurs in the mare and cattle. Although a clear increasing pattern in serum concentrations of estradiol-17[beta] and IGF-I was observed in camels with different ovulatory-sized follicles in the present study, this was not statistically significant due to the presence of the large standard deviations. The existence of a wide variation in blood steroid concentrations has been reported in the dromedary camel (11).

Our results showed that changes in the fine structure of the dromedary camel oocytes collected from preovulatory follicles are, in general, similar to that in cattle (12) and equine (13). Furthermore, the results of the present study revealed that oocytes from group 3 follicles showed more advanced signs of maturation as defined in the in vivo matured oocytes in cattle (12) and equine (13), and in vitro matured camel oocytes (7). The mean FF concentrations of estradiol and IGF-I were the highest in ovulatory-sized follicles (group 3) compared to the other follicle class size groups in the present study. Teissier (14) in the human, Grondahl (13) in the equine, Sirotkin (15) in the porcine and Zhao (16) in the rat reported that elevated estradiol in the follicular fluid indicate a more advanced stage of oocyte maturation.

We did not observe a massive formation of cortical granules in close proximity to the oolemma as the diameter of follicles increased. This was not similar to the well-defined dynamics of cortical granule formation in our previous observations during in vitro maturation in the dromedary camel oocyte (17). The reason for this different observation could be in vivo vs. in vitro environment, and or there may be more than one organelle involved in cortical granule formation (18). In the present study, in one oocyte from group 3, the first polar body had been fully extruded. This can be ascribed to the occurrence of an infertile mating and then surging in luteinizing hormone in days before slaughtering the animal. It has been shown that E/P ratio greater than 1 is a sign of healthy follicles and oocytes in cattle (19). Although E/P ratio decreased as the size of follicles increased in our study, however the ratio remained greater than 1. Considering the normal ultrastructure of the oocytes cytes and E/P ratio in the FF greater than 1 observed in the present study, it can be inferred that E/P ratio greater than 1 is an indicative for the healthiness of follicles in the dromedary camel.

Conclusion

In conclusion, the present study is the first to show that, the final stages of oocyte maturation in vivo is associated with increasing progesterone, estradio1-17[beta] and IGF-1 concentrations which are paralleled with ultrastructural changes leading to the final stages of oocyte maturation in the dromedary camel. Further, follicle diameter influences concentrations of progesterone, estradio1-17[beta] and IGF-I in the ET in the dromedary camel.

Acknowledgement

The authors would like to thank Professor M. Saeb for allowing us to perform hormone assay in his laboratory and Mr. A. Safavi for his technical assistance. The work was financially supported by Shiraz University. an.

* Corresponding author: Najmeh Davoodian, Ph.D., School of Veterinary Medicine, Shiraz University, Shiraz, Iran

E-mail: najmeh179@gmail.com

Received: 22 lune 2013

Accepted: 15 Sep 2013

References

(1.) Skidmore JA. Reproduction in dromedary camels: an update. Anim Reprod 2005:2(3):161-171.

(2.) Skidmore JA, Billah M. Allen WR. Investigation of factors affecting pregnancy rate after embryo transfer in the dromedary camel. Reprod Fert Devel 2002;14:109-1 16.

(3.) Badr MR, Abdel-Malak MG. In vitro fertilization and embryo production in dromedary camel using epididymal spermatozoa. Global Vet 2010;4:271-276.

(4.) Wani NA, Wernery U, Hassan FAH, Wernery R. Skidmore JA. Production of the first cloned camel by somatic cell nuclear transfer. Biol Reprod 2010;82(2):373-379.

(5.) Nili H, Mesbah F, Kafi M, Nasr Esfahani MH. Light and transmission electron microscopy of immature catnelus dromedarius oocyte. Anat Histo Embryol 2004;33(4): 196-199.

(6.) Davoodian N, Mesbah F, Kafi M. Oocyte ultrastructural characteristics in camel (Camelus dromedarius) primordial to large antral follicles. Anat Histo Embryo] 2011;40 (2): 120-127.

(7.) Kafi M, Mesbah F. Nil H, Khalili A. -Chronological and ultrastructural changes in camel (Camelus dromedarius) oocytes during in vitro maturation. Theriogenology 2005; 63(9):2458-2470.

(8.) Rahman ZU, Bukhari SA, Ahmad N, Akhtar N, Ijaz A, Yousaf MS, et al. Dynamics of follicular fluid in one-humped camel (Camelus dromedarius). Reprod Dom Anim 2008;43(6):664-671.

(9.) Spicer Li, Santiago CA, Davidson TR, Bridges TS., Chamberlain CS. Follicular fluid concentrations of free insulin-like growth factor (IGF)-I during follicular development in mares. Domest Anim Endocrinol 2005;29(4): 573-581.

(10.) Spicer LJ. Proteolytic degradation of Insulin-like growth factor binding proteins by ovarian follicles: A control mechanism for selection of dominant follicles. Biol Reprod 2004;70(5):1223-1230.

(11.) Skidmore JA. Reproductive physiology in female old world camelids. Anim Reprod Sci 2011;124(3-4):148-154.

(12.) Hyttel P. Callesen H, Greve T. Ultrastructural features of preovulatory oocyte maturation in superovulated cattle. J Reprod Fertil 1986;76:615-625.

(13.) Grondahl C, Hyttel P, Grondahl ML, Eriksen T, Gotered-sen P. Greve T. Structural and endocrine aspects of equine oocytes maturation in vivo. Mol Reprod Devel 1995;42:94-105.

(14.) Teissier MP, Chable H, Paulhac S. Aubard Y. Comparison of follicle steroidogenesis from normal and polycystic ovaries in women undergoing IVF: relationship between steroid concentrations, follicle size, oocyte quality and fecundabil ity. H urn Reprod 2000; 15(12):2471-2477.

(15.) Sirotkin AV, Dukesova J, Makarevich AV, Kubek A, Bulla J. Evidence that growth factors IGF-I, IGF-II and EGF can stimulate nuclear maturation of porcine oocytes via intracellular protein kinase A. Reprod Nutr Dev 2000; 40(6):559-569.

(16.) Zhao J, Taverne MA, Van der Weijden GC, Bevers MM, Van Den Hurk R. Insulin-like growth factor-1 (IGF-I) stimulates the development of cultured rat pre-antral follicles. Mol Reprod Dev 2001;58(3):287-296.

(17.) Revelli A, Delle Piane L. Casano S. Molinari E, Masso-brio M, Rinaudo P. Follicular fluid content and oocyte quality: from single biochemical markers to metabolom-ics: A review. Reprod Biol Endocrinol 2009;7:40-52.

(18.) Fair T, Hulshof SCJ, Hyttel P, Greve T, Boland M. Coo-cyte ultrastructure in bovine primordial to early tertiary follicles. Anat Embryol 1997;195:327-336.

(19.) Grado-Ahuir JA, Aad PY, Spicer U. New insights into the pathogenesis of cystic follicles in cattle: Microarray analysis of gene expression in granulosa cells. J Anim Sci 2011;89(6):1769-1786.

Mojtaba Kafi (1), Seyed Fakthroddin Mesbah (2), Najmeh Davoodian , and Ali Radivar (3)

(1.) Department of Anitnal Reproduction, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

(2.) Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

(3.) Research Institute of Animal Embryo Technology, Shahrekora' University, Shahrekord, Iran

Tablet. The mean[+ or -]SD follicular fluid and serum
concentrations of estradio1-17[beta], progesterone and IGF-I in
ovulatory-sized follicles in the dromedary camel

Groups  Follicle  No. of   Estradiol-17            Progesterone
        diameter  oocytes      [beta]               concentration
          (mm)               concentration              (ng/ml)
                                (ng/ml)

                                   FF         serum         FF

1        10-13.9        7     217.6[+ or  50.9[+  3.7[+ or -]0.7
                              -]25;5 (a)      or             (c)
                                          -]52.4

2        14-17,9        4     248.5[+ or  52.9[+       13.6[+ or
                                  -]14.9      or       -]3.4 (c)
                                          -]56.7

3          18-30        6     254.6[+ or  97.4[+       64.8[+ or
                              -]21.0 (b)      or      -]20.2 (d)
                                          -]77.5

            IGF-I                           FF
        concentration             Estrogen/Progesterone
           (ng/ml)                        ratio

scrum         FF          Serum

0.54[+     I40.1[+ or  153.2[+  59.07[+ or -]4.l2 (g)
    or     -]52.7 (C)       or
 -]0.7                  -]97.6

0.86[+     195.7[+ or  208.2[+   18.9[+ or -]5.19 (h)
    or     -]35.1 (c)       or
 -]0.8                  -]20.1

0.61[+     256.4[+ or  250,8[+   4.07[+ or -]1.34 (i)
    or     -]35.l (r)       or
 -]0.4                  -]89.4

Values in the same column with different superscripts
are significantly different a, b) significant at p<0..07;
c, d) significant at p<0.05; e, f) significant at p<0.01;
g, h, i) significant at p<0.001
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Title Annotation:Short Communication
Author:Kafi, Mojtaba; Falqhroddin Mesbah, Seyed; Najmeh; Davoodian, Ali Radivar
Publication:Avicenna Journal of Medical Biotechnology (AJMB)
Article Type:Report
Date:Jan 1, 2014
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