Effects of estradiol on in vitro maturation of buffalo and goat oocytes

Abstract Purpose The effects of estradiol on oocyte development seem to be varied among species. The present study investigated the effects of 17β‐estradiol on in vitro maturation of buffalo and goat oocytes. Methods Cumulus oocyte complexes (COCs) were aspirated from large antral follicles of slaughtered buffalo and goat ovaries. COCs were cultured in TCM‐199 medium supplemented with 0, 0.5, 1, and 1.5 µg/mL of 17β‐estradiol for in vitro maturation. Then, oocytes were used for the examination of state of nuclear maturation and cumulus expansion. Results In both species, oocytes treated with 17β‐estradiol showed higher cumulus expansion rate than control (0 µg/mL treated). In buffalo, the percentage of oocytes matured to the metaphase II (MII) stage increased in the concentration‐dependent manner of 17β‐estradiol. Similarly, estradiol positively influenced nuclear maturation of goat oocytes in vitro. Conclusions Estradiol has promoting effects on normalprogress of in vitro oocyte meiosis in buffalos and goats.


| INTRODUC TI ON
Assisted reproductive technologies (ARTs) can be applied to improve reproductive efficiencies in livestock species. In addition, ARTs have significant roles in the preservation of endangered species. 1 ARTs including artificial insemination (AI), cryopreservation, intra-cytoplasmic sperm injection (ICSI), multiple ovulation and embryo transfer (MOET), and in vitro embryo production (IVEP) have been used for improvement of domestic animals. In buffaloes, the response to superovulation treatment is poor, and ultimately, MOET is hardly applicable. However, oocytes recovered from slaughtered animals are used for IVEP. In vitro maturation (IVM) is the initial step of IVEP technology. IVM of oocytes involves collection of cumulus oocyte complexes (COCs) from antral follicles and culturing them until they reach to the metaphase II (MII) stage. 2 The success in IVM of oocytes depends on many factors including the size of follicles and oocytes, 3 the maturation environment, 4,5 and the culture media used. [6][7][8] Steroids influence oocyte maturation in vitro. 9 Estradiol is a major female steroid hormone, produced from cholesterol. To improve the nuclear and cytoplasmic maturation of oocytes as well as the expansion of the cumulus cells, gonadotrophin hormones and estradiol are usually used in IVM of buffalo 10 and caprine 11 oocytes. Although the estradiol is supplemented occasionally in IVM, its role in IVM of oocytes remains contentious. Estradiol has been shown to increase IVM of bovine oocytes 12,13 but Gliedt and colleagues have reported that estradiol negatively regulates cumulus expansion and in vitro embryo production in bovine. 14 It has also been reported that estradiol reduces the maturation of mouse oocytes 15,16 and that in vitro maturation of pig oocytes has been inhibited by estradiol. 17 A high concentration of estradiol (100 µg/mL) has also inhibited oocyte maturation in boer goats. 18 High concentration of estradiol inhibits spindle formation and polar body extrusion in bovine oocytes. 19 The effects of estrogen on oocyte maturation, ovulation, and embryo development seem to be species-dependent. 20 Its role is still unclear in some mammalian species.
Black Bengal goats are important genetic resource, available in Bangladesh and India. They are dwarf in size and popular for delicious meat with high marbling, high prolificacy, disease resistance, and adaptability in high temperature and humidity. Similarly, buffalo is economically an important species in livestock agriculture in Asia. They are popular for their high butter fat content in their milk, high feed conversion efficiency, and high disease resistance.
In Bangladesh, buffaloes are non-descriptive and river type. A few reports are available on in vitro development of oocytes in Black Bengal goats and indigenous river buffaloes in Bangladesh.
The aim of this study was to examine the effects of estradiol on in vitro maturation of oocytes from river buffaloes and Black Bengal goats.

| Chemicals
Unless otherwise mentioned, all chemicals were purchased from Sigma-Aldrich (St. Louis).

| Collection and processing of ovaries
Ovaries were collected from indigenous river buffaloes and Black Bengal goats at local slaughter house and kept in a thermo flask containing 0.9% physiological saline for transportation to the laboratory. The ovaries were washed in Dulbecco's phosphate buffer saline (DPBS) solution supplemented with gentamycin (50 µg/mL) once and following three times in DPBS. After trimming surrounding tissues, ovaries were washed again with saline solution.

| Collection of COCs
The collection and in vitro maturation of cumulus oocyte complexes (COCs) were done according to Totey et al 21

| In vitro maturation of COCs
The maturation medium was prepared with TCM-199 supplemented with 0.1 mg/mL sodium pyruvate, 0.08 mg/mL gentamycin sulfate, 5% (v/v) fetal bovine serum (FBS), and 100 ng/mL follicle-stimulating hormone (FSH). 22 In order to investigate the effects of 17β-estradiol, maturation medium was also supplemented with 0, 0.5, 1.0, or 1.5 µg/mL of 17β-estradiol. COCs were placed in 50 µL maturation droplets of maturation medium under paraffin oil. Each droplet was placed in a separate culture dish (No. 430165, 35 mm cell culture dish, Corning Incorporated). They were cultured for 24 hours at 38.5°C temperature with 5% CO 2 in humidified air.

| Assessment of cumulus expansion and meiotic stage of oocytes
After in vitro maturation, the assessment of cumulus expansion was carried out as described by Maruska  The stained oocytes were classified on the basis of morphology of the chromatin and nuclear envelope. [24][25][26][27] Oocytes after resumption of meiosis, the stages were classified into early diakinesis (ED), late diakinesis (LD), metaphase I (MI) and metaphase II (MII).
Oocytes showing cytoplasmic or nuclear abnormalities were considered degenerated oocytes.

| Data presentation and statistical analysis
All data from cumulus expansion and meiotic stage assessment experiments were subjected to one-way ANOVA followed by Tukey's HSD test (IBM SPSS Statistics 22). Differences at P < .05 were considered statistically significant.

| Effect of 17β-estradiol on cumulus expansion during in vitro maturation of COCs
The typical morphologies of COCs before and after in vitro maturation in the medium supplemented with 0, 0.5, 1, and 1.5 µg/mL of 17β-estradiol in buffaloes and goats are shown in Figures 1 and   2, respectively. Assessment of oocyte maturation by visualization of cumulus expansion of COCs revealed a significant difference in expansion rate due to estradiol supplementation in both buffaloes and goats. In buffaloes, supplementation of in vitro maturation medium with 17β-estradiol (0.5, 1.0 and 1.5 µg/mL) significantly increased cumulus expansion rate (93%, 100%, and 100%, respectively) compared to control ( Figure 3A). In goats, supplementation of medium with 1 µg/mL 17β-estradiol significantly increased cumulus expansion rate (100%) compared to 0, 0.5, and 1.5 µg/mL groups (73%, 80%, and 70%, respectively) ( Figure 3B).

| Effect of 17β-estradiol on nuclear maturation of buffalo and goat oocytes
The representative images of nuclear morphology of buffalo and goat oocytes after maturation are shown in Figure 4. After resumption of meiosis, few proportion of buffalo oocytes were at early diakinesis in 0.5 µg/mL (10%) and 1 µg/mL (5%) of 17β-estradiol supplemented groups ( Figure 5A). The proportions of buffalo oocytes at late diakinesis increased with increment of 17β-estradiol concentration. The percentages of MII oocytes increased in concentrationdependent manner with 17β-estradiol treatments.

| D ISCUSS I ON
The rates of in vitro maturation of mammalian oocytes including mice, cattle, and sheep are low compared with oocytes matured in vivo. [28][29][30] This might be due to poor nuclear maturation caused by inadequate culture conditions. 31-34 Several factors are included in culture media to improve the success rate of in vitro maturation.
Here, we found that estradiol increased the percentage of MII oocytes. It is thought that estradiol supports nuclear maturation of oocytes during in vitro maturation. It has been reported that estradiol increases nuclear maturation of human oocytes. 35  Here, higher numbers of buffalo oocytes treated with 1.5 μg/mL of estradiol reached the MII stage than other groups. In goat, the number of oocytes at the MII stage significantly increased in 1 μg/ mL estradiol but decreased with 1.5 μg/mL of estradiol, which further indicated that high concentration of estradiol inhibited meiotic maturation of goat oocytes. Previously, it was reported that a proportion of estradiol was absorbed by paraffin oil covering the microdrops of culture medium. 62 In the present study, each droplet was placed in a separate culture dish and, thus, estradiol could not move F I G U R E 4 Nuclear morphologies of buffalo and goat oocytes after in vitro maturation. COCs were cultured in medium supplemented with 0, 0.5, 1, and 1.5 µg/mL of 17β-estradiol during in vitro maturation. After maturation, oocytes were denuded mechanically, fixed with aceto-ethanol, stained with aceto-orcein, and examined for nuclear maturation. Scale bar represents 10 μm from one droplet to another. It was considered that estradiol was equally absorbed from each droplet without affecting the estradiol concentration.
In conclusion, estradiol enhances nuclear maturation of oocytes in both buffaloes and goats.

ACK N OWLED G EM ENTS
We are thankful to Steve Clark, Senior Research Scientist (Retired), Agriculture Victoria, Australia, for his critical suggestions for improvement of this manuscript. We are also grateful to the staff of local slaughter house for providing us ovaries.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

H U M A N/A N I M A L R I G HTS
This article does not contain any studies with human and animal subjects performed by the any of the authors.