Effect of LH and Estradiol-17β Supplementation at Different Time Points on In vitro Development of Preantral Follicles in Sheep

It is known that mammalian ovaries contain thousands to lakhs of oocytes enclosed in the primordial follicles. But less than one percent of the primordial follicles grow to become graafian follicles and ovulate. The remaining 99.9 percent primordial follicles never reach ovulation and they degenerate through a process known as atresia. This fact has stimulated great interest in the development of a culture system that might be able to maintain follicular growth and avoid this loss of follicles (Araujo et al., 2014). It is necessary to develop culture strategies that support the activation and sustained in vitro growth of primordial follicles in order to maximize the reproductive potential of ovarian tissue (Picton et al., 2008). Accordingly several laboratories world over have been developing techniques to harvest, culture and produce embryos from the oocytes in preantral follicles (PFs’) in the ovaries of different species of laboratory and domestic animals. A developing biotechnology called in vitro follicle culture (IVFC) might increase the number of potentially fertilizable oocytes by recovering, in vitro culturing and preserving those follicles, for the purpose of prevention of atresia (de Figueiredo et al., 2011). Recent innovations to augment reproductive efficiency of female mammals such as superovulation, embryo transfer, in vitro embryo production, somatic cell cloning etc. are all oocyte intensive technologies. Such techniques could provide access to a new source of female germplasm and represent a huge opportunity even with a low over all success rate of just one percent since it would be possible to produce one thousand or more embryos from both the ovaries of a slaughtered female mammal.
       
Although the mechanisms regulating the activation and subsequent growth of primordial follicles remain poorly understood, accumulating evidence indicates that follicular development depends on the presence of hormones and growth factors. Ovarian follicular development and growth are controlled by many hormones and growth factors. Several publications have been documented for the effects of different hormones and growth factors include follicle stimulating hormone (FSH), luteinizing hormone (LH), growth hormone (GH), Thyroxine (T4), estradiol-17β, Insulin like growth factor - I (IGF-I), Vascular endothelial growth factor (VEGF), Epidermal growth factor (EGF) and Growth differentiation factor - 9 (GDF-9) supplementation in culture medium on in vitro development of PFs’ in sheep and goat (Martins et al., 2010, Andrade et al., 2011, Arunakumari et al., 2013, Costa et al., 2014, Silva et al., 2015, Lima et al., 2016, Cadenas et al., 2017 and Monte et al., 2019).
       
The glycoprotein hormone, LH may play multiple roles throughout follicular development. The addition of LH is required for in vitro development of preantral follicles to the antral stage in rats (Wu et al., 2000). The follicle survival rate was significantly higher in medium supplemented with LH (Park et al., 2013b). Several in vitro studies have suggested that the development of early follicles occur under the influence of gonadotropins (Cortvrindt et al., 1998a, b; Wu et al., 2000). LH receptors first develop on the cells of theca interna at the preantral stage of development (Richards et al., 1995) and the granulosa cells of large antral follicles (Garverick et al., 2002).
       
Estradiol is a hormone synthesized by aromatase, present in the granulosa cells and its production depends on gonadotrophin secretion (Nelson and Bulun, 2001). Granulosa cells that are derived from early antral follicles secret estradiol and are involved in antral formation. It is indeed known that estradiol can act as an inhibitor of the apoptosis induced by oxidative stress in luteal and follicular cells (Murdoch, 1998). Estradiol may act to support granulosa cell differentiation, after which these cells become receptive to gonadotrophin. Estradiol typically acts through specific intracellular receptors, ERα and ERβ, which are expressed in granulosa cells of preantral and antral follicles (Tomic et al., 2007).
       
Although LH and estradiol-17β have been used for the in vitro culture of preantral follicles yet, the suitable time points of supplementation of LH and estradiol-17β is not known. Therefore, the aim of present study was to evaluate the influence of addition of LH and estradiol-17β on in vitro development of preantral follicles (PFs’) in sheep and to study the effect of LH and estradiol-17β on development of preantral follicles (PFs’) in sheep at different time intervals.

Chemicals and media
 
The chemicals and reagents used in this study were obtained from Sigma Chemical Co. (USA) unless otherwise indicated.
 
Collection of ovaries, isolation, selection and culture of preantral follicles
 
Sheep ovaries (n=120) were collected irrespective of age, body condition and season at a local slaughter house for a total of ten repetitions. Immediately after slaughter, the ovaries were washed in 70% ethyl alcohol followed by two washes in Bicarbonate buffered tissue culture medium 199 (TCM 199B) supplemented with gentamycin (50 µl/ml). The ovaries were transported to the Embryo-biotechnology laboratory, Department of Veterinary Physiology, College of Veterinary Science, Tirupati  in TCM 199B within 1 h at 4°C.   
       
Preantral follicles were isolated as per the methods standardized in the laboratory. In brief fat and connective tissue surrounding the ovaries were removed. The ovarian cortex was cut into thin slices (1-2 mm thick) from which intact preantral follicles in the size range of 250 - 400 µm were mechanically isolated without damaging to the basement membrane by micro-dissection method under stereo zoom microscope as represented in Fig 1. Total 9 treatment groups (T1 to T9) as represented in Table 1.
 

 

       
Standard medium was prepared by supplementing Bicarbonate buffered tissue culture medium 199 (TCM 199B) with Gentamycin (50 µl/ml); T₄ (100µl/ml); FSH (175µl/ml); IGF-1 (200 µl/ml); GH (200 µl/ml) as represented in Table 2 which supported the best development in vitro of sheep preantral follicles earlier (Arunakumari et al., 2010).
 

       
Preantral follicles were cultured in micro drops of TCM 199B / Standard medium supplemented variously with LH (2 μg/ml) and estradiol-17β (5 ng/ml) for a period of six days. Follicles in culture were morphologically evaluated every 24h for the proportions exhibiting growth, average increase in diameter and proportion of PFs’ exhibiting antrum formation. Cumulus Oocyte complexes (COCs) were collected at the end of six day culture and subjected to additional 24h of in vitro maturation. Meiotic maturation to MII stage was assessed by staining the oocytes with Hoechst 33342 medium.
 
Statistical analysis
 
Statistical analysis of the data was analyzed by one way ANOVA by using SPSS 20 software. Duncan’s multiple range test (DMRT) and percentile deviation tests were applied to identify the significance of difference among different treatment groups.

Supplementation of luteinizing hormone (LH) and estradiol-17β at different time points during in vitro culture of sheep PFs’ are shown in Table 3 and Fig 1 to Fig 4. The present study demonstrated the importance of LH and estradiol-17β for growth, activation and maturation of sheep preantral follicles. In our present experiment supplementation of LH and estradiol-17β stimulates in vitro growth of sheep PFs’. It is interesting to note that LH and estradiol-17β supplementation during last two days (T7) of the culture supported better proportion of PFs’ exhibiting growth whereas supplementation of LH and estradiol-17β during first two days (T5) of the culture supported better average increase in diameter and proportion of PFs’ exhibiting antrum formation at the end of six day culture and the oocytes in COCs isolated at the end of culture in these treatments and subsequently subjected to in vitro maturation (IVM) for 24hr developed (T5 and T7) at a higher frequency to MII (metaphase II) stage.
 

 

 

 

 

       
Supplementation of LH and estradiol-17β hormones during initial stages of the culture maintain cell to cell contact viz., oocyte granulosa cell interaction and avoid degeneration therefore leading to better development of PFs’ in vitro. Therefore, in the present study it is possible that the combination of hormones significantly influenced the growth of preantral follicles and this may be the effect of increased cell mass through granulosa and theca cells proliferation that have contributed to the proportion of preantral follicles exhibiting growth, average increase in follicular diameter and also antrum development together with increase in endothelial cell proliferation and vasculature surrounding the follicle.   
       
From the present results it appears that inclusion of LH and estradiol-17β during the first two days of culture supports better average increase in diameter (T5 in Fig 2), antrum formation (T5 in Fig 3) of PFs’ and maturation of oocytes to MII stage (T5 in Fig 4) in sheep, but inclusion of LH and estradiol-17β during last two days of culture (T7 in Fig 1) supports better proportion of preantral follicles exhibiting growth. Similar results reported by Flaws et al., (1997), they observed increase in diameter and growth of primordial follicles with high levels of LH supplementation, it also facilitates the transition to the primary and secondary follicle stage. A higher rate of primordial follicle activation was recorded in the ovarian cortex cultured in medium supplemented with high concentrations of LH, after 7 days of culture LH increased the follicular diameter. LH receptors are expressed in the interstitial cells, as well as the theca and granulosa cells. These cells are probably stimulated by LH and then secret factors that promote preantral follicular growth (Saraiva et al., 2008). To support this hypothesis, Liu et al., (2002) demonstrated that in the absence of LH, no proliferation of granulosa cells could be observed in vitro. These findings suggest that LH promotes follicular development, especially during the preantral, early antral follicle transition, by up-regulating follicular androgen biosynthesis (Orisaka et al., 2013).
       
The interest of studying the effects of LH in in vitro maturation is obvious, as this hormone plays a fundamental role in androgen production by theca cells from the earliest stages of follicle growth (Richards et al., 1986). LH plays a key role in stimulating the enzymes responsible for androgen production in the theca cells and in initiating the final differentiation of the GCs. Recent immunohistochemical studies have demonstrated that LH receptor is also initially expressed in cumulus cells during follicular development, suggesting that LH might interfere during the oocytes entire growth phase. It is known that gonadotropins stimulate the production of anti-apoptotic proteins and therefore stimulate follicular survival (Markstrom et al., 2002).
       
Investigation of the influence of LH on follicle maturation in vitro showed that follicle survival rate was significantly higher in medium supplemented with LH than without (Park et al., 2013a). Moreover, LH bioactivity had an effect on antral cavity formation (Cortvrindt et al., 1998b). It has been suggested that LH supplementation during primary and secondary follicle culture enables follicles to respond to later LH-dependent growth (Wu et al., 2000). In previous reports of follicle culture, LH supplementation did not affect follicle survival, but did enhance follicle growth and antrum formation in the mouse and human, increasing the rate of oocyte maturation to the metaphase II stage in mice (Abir et al., 1997; Cortvrindt et al., 1998a) and Cortvrindt et al., (1998b) found that LH generated favorable conditions for the transition of oocytes from metaphase I to metaphase II in mice.
       
Although there are few studies on the influence of estradiol on growth, antrum formation and maturation, it is known that estradiol can act as an inhibitor of the apoptosis in luteal and follicular cells (Murdoch, 1998). Zheng et al., (2003) observed oocyte developmental capacity during IVM in rhesus monkey was stimulated by supplementation of 17β estradiol. Similar results were reported by Tasaki et al., (2013), they observed highest rate of antrum formation with supplementation of culture medium with estradiol and no antrum formation without estradiol in PFs’ of pig. Proper 17β estradiol levels promote follicle growth and inhibit follicle atresia. 17β estradiol was thought to exert its role in promoting folliculogenesis by favoring granulosa cell proliferation and inhibiting apoptosis signals. Endo et al., (2013) observed supplementation of the culture medium with estradiol improved the cavity formation and the highest ratio of antrum cavity formation was obtained with the medium containing estradiol-17β. Various studies have reported that 17β estradiol favors cell proliferation and survival by activating the transcription of factors required for progression through the cell cycle, while repressing others that cause cell cycle arrest and apoptosis (Craig et al., 2014).
       
The result of our study, in which supplementation of LH and estradiol-17β to TCM 199B culture medium in early stages followed by standard medium alone in later stages supports better development of PFs’ in vitro. Similarly, supplementation of estradiol with gonadotropins promote caprine primordial follicle activation and in vitro follicular growth (Lima-Verde et al., 2010). Therefore, LH and estradiol-17β may play an important role in the process of preantral follicular development and maturation.

It was concluded that the supplementation of LH and estradiol-17β during first two days (0-2 days) of the culture followed by standard medium alone in later stages (3-6 days) supported better average increase in diameter and proportion of PFs’ exhibiting antrum formation at the end of six day culture. Further the oocytes in COCs isolated at the end of culture in these treatments and subsequently subjected to in vitro maturation (IVM) for 24hr developed at a higher frequency to MII (metaphase II) stage. Supplementation of LH and estradiol-17β to TCM 199B culture medium during last two days (5-6 days) of the culture supported better proportion of PFs’ exhibiting growth. Following supplementation with LH and estradiol-17β for the first two days culture of PFs’ in standard medium appears to be advantageous for the development of preantral follicles in vitro.