Kisspeptin stimulates sheep ovarian follicular development in vitro through homologous receptors

Summary

The present study was conducted to elucidate (1) the influence of kisspeptin (KP) on the in vitro development of preantral follicles (PFs) and (2) evolution of KP receptor gene (KISS1R) expression during ovarian follicular development in sheep. Kisspeptin was supplemented (0–100 µg/ml) in the culture medium of PFs for 6 days. The cumulus–oocyte complexes (COCs) from cultured PFs were subsequently matured to metaphase II (MII) for an additional 24 h. The proportions of PFs exhibiting growth, antrum formation, average increase in diameter, and maturation of oocytes to MII stage were the indicators of follicular development in vitro. The expression of the kisspeptin receptor gene at each development stages of in vivo developed (preantral, early antral, antral, large antral and COCs from Graafian follicles) and in vitro cultured PFs supplemented with KP was assessed using a real-time polymerase chain reaction. The best development in all the parameters under study was elicited with 10 µg/ml of KP. Supplementation of KP (10 µg/ml) in a medium containing other growth factors (insulin-like growth factor-I) and hormones (growth hormone, thyroxine, follicle-stimulating hormone) resulted in better PF development. The KISS1R gene was expressed in follicular cells and oocytes at all the development stages of both in vivo developed and in vitro cultured follicles. Higher KISS1R gene expression was supported by culture medium containing KP along with other hormones and growth factors. Accordingly, it is suggested that one of the mechanisms through which KP and other growth factors and hormones influence the ovarian follicular development in mammals is through the upregulation of expression of the KP receptor gene.

Introduction

The frequency of in vitro embryogenesis from cultured preantral follicles (PFs) is relatively poor (Arunakumari et al., 2010). Efforts to improve culture conditions often involve supplementation of culture medium with different hormones and growth factors (Kona et al., 2016). In this connection, kisspeptin a peptide hormone (145 amino acids), a product of the KISS1 gene, synthesized and released from arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV) of hypothalamus is an interesting but inadequately investigated growth factor. Kisspeptin was reported to be involved in upstream regulation of pulsatile and surge GnRH release, sex differentiation in the brain, gonadotropin secretion, onset of puberty, ovulation, metabolic regulation of fertility (Roa et al., 2008a; Pinna et al., 2012), seasonal reproduction, follicular development (Fernandois et al., 2017), oocyte maturation (Lapatto et al., 2007), implantation, and gonadal steroid feedback to the hypothalamus (Horikoshi et al., 2003). However, the influence KP on the development of cultured PFs in sheep was never investigated.

Kisspeptin receptor (KISS1R) belongs to rhodopsin gamma family of G-protein coupled receptors and was identified for the first time as an orphan receptor in the rat brain (Lee et al., 1999). The expression of KISS1R mRNA and its immunohistochemical localization in the ovaries of mice (Laoharatchatathanin et al., 2015; Merhi et al., 2016), hamster (Shahed and Young, 2009), rabbit (Maranesi et al., 2019), cat (Cielesh et al., 2017), dog (Tanyapanyachon et al., 2018), swine (Saadeldin et al., 2012), goat (Inoue et al., 2009), bovine (Mishra et al., 2019), and human (Gaytán et al., 2009; García-Ortega et al., 2014; Merhi et al., 2016; Blasco et al., 2019; Blasco et al., 2020) suggest that kisspeptin (KP) may have direct gonadal effects (Clarke et al., 2015). However, the evolution of KISS1R expression as the ovarian follicles and oocytes develop from preantral to Graafian follicle stage has not been reported previously. Furthermore it is unknown whether in vitro culture of ovarian follicles in mammals provides for the expression kisspeptin receptor.

In view of the above, the present study was conducted in sheep to (1) know the influence of kisspeptin on the development of cultured PFs, (2) evaluate the interaction of kisspeptin with other hormones and growth factors on in vitro growth of PFs, and (3) elucidate the expression of the KISS1R gene as the ovarian PFs developed to the Graafian follicle stage in in vivo developed and cultured PFs in vitro.

Materials and methods

All the methods described briefly here are routinely used in the culture of PFs and described in detail in several earlier publications from our laboratory (Arunakumari et al., 2013; Kona et al., 2016; Srividya et al., 2017; Kumar et al., 2019; Kona et al., 2021). Unless otherwise stated, culture medium, hormones, growth factors, fetal calf serum and all the other chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and plastics from Nunclon (Roskilde, Denmark). All the hormones and growth factors used were cell culture tested and endotoxin free.

Collection, processing and isolation of different categories of follicles (PFs) from sheep ovaries

In total, 1148 ovaries recovered immediately after slaughter on different days were transported to the laboratory within 1 h in sterile, warm (37°C) phosphate-buffered saline. In total, 6592 intact PFs) isolated from the collected ovaries in the size range 250–400 µm (standardized in our laboratory and quoted in several earlier publications from our laboratory; Arunakumari et al., 2013; Kona et al., 2016; Kona et al., 2021; Srividya et al., 2017; Kumar et al., 2019) were used in the study. PFs having visible centrally placed oocytes without any signs of atresia and with intact basement membrane and no antral cavity were considered to be good for the culture (Figure 1B, C). On average, the diameter of the PFs on day 0 was 321.3 ± 6.55 µm. In the size range of the follicles used here, a small proportion that may have initiated antrum formation were eliminated from in vitro culture. The PFs were cultured for 6 days in different groups (Tables 1–5) with subsequent in vitro maturation of the cumulus–oocytes complex (COC) for an additional 24 h, following methods developed in the laboratory (Arunakumari et al., 2013; Kona et al., 2016, 2021; Srividya et al., 2017; Kumar et al., 2019). These procedures are briefly mentioned in subsequent sections.

Figure 1. Different stages of in vitro and in vivo grown ovarian follicles used in the present studies. (A) Primordial follicles. (B, C) Preantral follicles at isolation 250–400 μm in diameter. (D) Early antral follicles. (E) Preantral follicles cultured for 2 days. (F) Antral follicles. (G) Preantral follicles cultured for 4 days. (H) Graafian follicle. (I) Preantral follicles cultured for 6 days. (J, K) Cumulus–oocyte complex from in vivo and in vitro cultured follicles. (L, M) Oocyte freed from follicular cells of (L) and (M) for the MII stage.

Table 1. Effect of kisspeptin (0–100 µg/ml) on in vitro development of sheep preantral follicles (PFs). Each replicate: five preantral follicles; no. of replicates/group = 10

Control medium: TCM199B medium containing 50 µg/ml gentamycin sulphate.

Values with different superscripts within a column are significantly different (P ≤ 0.05).

Table 2. Effect of kisspeptin (10–20 µg/ml) on in vitro development of sheep preantral follicles (PFs). Each replicate: five preantral follicles; no. of replicates/group = 10

Control medium: TCM199B medium containing 50 µg/ml gentamycin sulphate.

Values with different superscripts with in a column are significantly different (P ≤ 0.05).

Table 3. Effect of 10 µg/ml of kisspeptin in combination with standard medium (SM) on in vitro development of sheep preantral follicles (PFs). Each replicate: five preantral follicles; and no. of replicates/group = 10

Control medium: TCM199B medium containing 50 µg/ml gentamycin sulphate.

Standard medium composition (SM).

¹ TCM199B containing 1 µg/ml thyroxine, 2.5 µg/ml FSH, 10 ng/ml insulin-like growth factor-I and 1 mIU/ml of growth hormone.

Values with different superscripts within a column are significantly different (P ≤ 0.05).

Table 4. Design for KISS1R gene expression studies in in vivo developed and in vitro cultured PFs in sheep

For gene expression each sample of total RNA isolation from cumulus cells and oocytes, 50–60 cultured PFs were used for each respective culture duration of 20 stages. As the quantity of RNA acquired in the follicular cells is low, the RNA isolation was repeated four or five times for every stage, therefore almost 5082 follicles were used in the total RNA isolation.

Table 5. Expression of kisspeptin receptor (KISS1R) gene in follicular cells and oocytes in in vivo developed and in vitro cultured PFs follicles

Values with different superscripts within a column are significantly different (depicted with a, b, c, d, e).

Values with different superscripts within a column are significantly different (depicted with 1, 2, 3, 4, 5).

Culture of PFs, in vitro maturation of COCs and evaluation

Preantral follicles were cultured in bicarbonate-buffered tissue culture medium 199 (TCM199B) supplemented with 50 mg/ml gentamycin sulphate and different concentrations of kisspeptin-10 (KP; M 2816; Sigma-Aldrich, USA; Tables 1 and 2) and/or other growth factors and hormones (Table 3). Culture medium was pre-incubated for 1 h at 39°C under an humidified atmosphere of 5% CO₂ in air. The selected follicles were washed three times in culture medium and subsequently placed individually in 20-µl droplets of culture medium in 35-mm plastic culture dishes (cat. no. 153066, Nalge Nunc, Denmark). To avoid evaporation of the medium, the microdroplets were overlaid with autoclaved lightweight mineral oil (Sigma M8410) pre-equilibrated with the medium overnight at 39°C in 5% CO₂ in air. These culture dishes were incubated at 39°C under an humidified atmosphere in 5% CO₂ in air for up to 6 days. The day on which the PFs were placed in the culture was designated as day 0 and the subsequent days as day 1, day 2 and so on. Half the medium was replaced by an equal volume of fresh medium every 48 h.

Each follicle was evaluated morphologically every 24 h during the culture period using an inverted microscope (Leica, DMIRB, Germany) for increase in the diameter and antrum formation. The average increase in diameter was calculated only for the growing follicles during the culture period in all the groups under study.

After 6 days of culture, the cumulus–oocyte complexes (COCs) were isolated by gently opening the cultured follicles (Figure 1J, K) mechanically with a 24G needle under a zoom stereomicroscope. The isolated COCs (Figure 1J, K) were subjected to further in vitro maturation for 24 h. The COCs were washed three times in in vitro maturation (IVM) medium (d’Anglemont de Tassigny et al., 2008; TCM199B supplemented with 10 µg/ml follicle-stimulating hormone (FSH), 10 µg/ml luteinizing hormone, 1 µg/ml estradiol-17β, 50 µg/ml gentamycin sulphate, 10 µg/ml bovine serum albumin (BSA; A8412, Sigma, USA) and 10% (v/v) oestrous sheep serum. After washing the COCs were placed individually in 20-µl droplets of the same medium in 35-mm plastic culture dishes, covered with pre-equilibrated lightweight mineral oil, and incubated for 24 h as described above.

After IVM, the COCs were subjected to repeat pipetting through a fine bore glass pipette during which the oocytes were denuded of cumulus cells. Subsequently the oocytes were washed in Hoechst 33342 fluorescent stain solution (B2261; concentration of 5 µg /ml) and incubated in a 50-µl droplet of the same solution for 15 min at 39°C (d’Anglemont de Tassigny et al., 2008) and examined under fluorescent light (510–560 nm wavelength) on an inverted microscope (Leica, Germany; excitation: 352–455 nm and emission 460–490 nm) for the MII stage (Figure 1L, M).

Quantitative expression of the KISS1R gene

Quantitative expression of the KISS1R gene was studied from the preantral follicle stage onwards in the in vivo developed and corresponding stages from four groups of cultured ovarian follicles (Table 4; see Supplementary Table and Figure 1 for equivalent stages between in vivo developed and in vitro cultured follicles). The entire experiment was repeated four times. Triplicate samples of complementary DNA (cDNA) from each replicate of the experiment (4 × 3 = 12 cDNA) samples for each in vivo and in vitro stage were subjected to reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Isolation and quantification of total RNA from follicular cells and oocytes in each group

At each of the development stages of follicles (from PFs stage onwards) from in vivo developed and the cultured follicles (Table 4), follicular cells and oocytes were separately pooled from 30–50 follicles collected on the same day from the ovaries for the isolation of total RNA. RNA isolation was carried out using patented Medox-Easy spin column Total RNA Mini prep Kits (Srividya et al., 2017; Kumar et al., 2019) (26–27) according to the manufacturer’s instructions (Medox Biotech India Pvt. Ltd, Chennai, India). The RNA concentration and purity were determined using a NanoDrop lite spectrophotometer (Thermo Scientific S. No.1354). RNA samples with purity (absorbance at 260/280) only in the 1.8–2.1 range were used in the expression studies. The RNA sample was stored at −70°C until analyzed.

Reverse transcription and real-time PCR

High-capacity reverse transcription kit (Applied Biosystems part no. 4368814) was used for the reverse transcription. RT reaction was carried out for 10 min at 25°C, for 120 min at 37°C and for 5 min at 85°C in a thermocycler (Eppendorf Mastercycler Gradient) according to the manufacturer’s instructions. In a comparison of 12 commonly used reference genes, RPLPO, HPRT1 and 18S rRNA were the three most stably expressed genes in sheep ovarian follicles under the current experimental conditions (unpublished observations in our laboratory). Therefore, the geometric mean of these three genes (Khamsi and Roberge, 2001) was used as the normalizer in the analysis of the expression of kisspeptin receptor gene. Primer and probe details for kisspeptin receptor and the reference genes are given in Table 6. Real-time RT-qPCR was performed on an Applied Biosystems 7500 machine. Each 25-µl reaction mix contained 12.5 µl of TaqMan Universal PCR Master Mix (2×), 1.25 µl of 20× gene expression assay mixture, and 25 ng of cDNA sample in nuclease-free water. Thermal cycling conditions were Erase UNG (uracil N-glycosylase) activation 2 min at 50°C, AmpliTaq Gold DNA polymerase activation10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Extreme Ct (threshold cycle number) values and ‘no detection’ found for some of the samples meant that these were discarded prior to calculation of relative quantification (RQ) values, resulting in unequal numbers of observations in different groups. To calculate the expression levels (RQ values) of different target genes, first the Ct values of the target and reference genes were converted to quantity inputs using the formula 2^{minimumCt − sampleCt}. Expression of the target genes was the ratio of target quantity input to that of geometric mean of quantity inputs of the reference genes.

Table 6. Primers and probe sequences of target gene (kisspeptin receptor, KISS1R) and reference genes

Experiments

In total, four experiments were conducted to study the influence of KP on in vitro development of PFs and KISS1R gene expression in sheep.

Experiment 1

Experiment 1 was initially conducted with KP (concentration ranges 1, 5, 10, 15, 20, 25, 50 and 100 µg/ml) to ascertain whether KP could influence the in vitro development of PFs (Table 1)

Experiment 2

Based on the results of Experiment 1, and having confirmed in Experiment 1 that KP could influence the development of PFs, Experiment 2 was conducted to determine the best concentration for KP for PFs development in in vitro culture (Table 2).

Experiment 3

It was undertaken to discover whether KP at 10 µg/ml (as determined from Experiment 2) interacted with other hormones and growth factors [standard medium (SM)] on the in vitro development of sheep PFs (Table 3).

Experiment 4

This experiment was conducted to elucidate whether observed improvement of KP on in vitro development of PFs was due to the expression of its receptor through gene expression using RT-PCR studies (Tables 4 and 5). Receptor expression was studied in in vitro cultured follicles and compared with the in vivo developed PFs

Statistical analysis

Dependent variables are the development parameters of the follicles, and independent variables are the different treatments in the experiments. A comparison of the proportion of PFs exhibiting growth, average increase in diameter, and antrum formation in the in vitro cultured PFs among different treatment groups was undertaken separately using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test using SPSS 20 software. The meiotic maturation of the oocytes to MII stage was calculated using simple percentage analysis at the end of the culture period. For gene expression studies, the stage of development and source (in vivo or in vitro) were the independent variables, and expression of the genes was the dependent variable. Log RQ values were analyzed using two-way ANOVA [General Linear Model (GLM)] with unequal numbers of observations followed by the Tukey honestly significant difference (HSD) multiple comparison test (SPSS version 20, IBM Corp., USA). A P-value ≤ 0.05 was considered statistically significant.

Results

Influence of kisspeptin on in vitro growth of PFs in sheep

The influence of KP on preantral follicular development is shown in Tables 1 and 2. It was observed that KP at 10 µg/ml supported a significantly better development of PFs (Tables 1 and 2) compared with all other concentrations for all the parameters studied in both Experiments 1 and 2. Furthermore the SM (growth factors and hormones) supplemented with KP 10 µg/ml supported the best development of PFs in vitro (Table 3). Therefore 10 µg/ml of KP was subsequently used in the KISS1R gene expression studies.

Quantitative expression of KISS1R gene in in vitro developed ovarian follicles and in vitro cultured PFs in sheep

Expression of kisspeptin receptor gene in follicular cells and oocytes from in vivo developed ovarian follicles and in vitro cultured PFs

While KISS1R expression in follicular cells was quantitatively different among different development stages of in vivo developed and in vitro cultured follicles, it increased as the development progressed in both groups (Table 5). While the follicular (cumulus cells) cells in in vitro matured COCs from the follicles cultured in Groups 3 and 4 exhibited a significant increase in the expression (Table 5), the follicular cells (cumulus cells) in in vitro matured COCs from the follicles cultured in Groups 1 and 2 exhibited a significant drop in expression (Table 5). Furthermore the development from the early antral to the antral stage marked the transition to better expression of KISS1R in the follicular cells among all groups of follicles either in vivo developed or in vitro cultured (Table 5).

The expression patterns of KISS1R in the oocytes from different development stages of in vivo developed and in vitro cultured follicles was parallel to that observed in the follicular cells (Table 5), increasing as development progressed from the preantral stage. Similar to the follicular cells, the transition to better expression occurred as the follicles developed to the antral stage (Table 5). However, unlike the follicular cells, a significant drop in the expression of KISS1R was observed in the oocytes cultured in Groups 1–3 media as they developed from the antral to the large antral stage (Table 5). The pattern of gene expression of KISS1R in oocytes as observed in in vivo was mimicked at all development stages in the oocytes from the follicles cultured in Group 4 (Table 5)

Discussion

This is the first study to examine the direct effects of kisspeptin (KP) on the in vitro development of PFs in sheep. It was observed in this study that KP at lower concentrations promoted the growth of PFs and yielded better results compared with that of higher concentrations (Table 1). As we investigated a reasonably large number of different concentrations, it may be suggested that 10 µg/ml appears to be the best, if not optimum, for the in vitro development of sheep PFs (Tables 1 and 2). It must be pointed out that the KP concentration that supported follicular growth in vivo might be higher, as suggested by the high concentrations of KP in the follicular fluid compared with serum in the pig (Saadeldin et al., 2012) and women (Singh et al., 2021). However, intrafollicular concentrations of KP in sheep are unknown.

The detrimental effects of higher doses of kisspeptin on follicular growth in vitro have also been reported earlier (Mamo et al., 2007; Byri et al., 2017; Fernandois et al., 2017). In a study in goats, supplementation of kisspeptin at 1 and 10 µM concentrations facilitated the development of primordial follicles, while higher doses of 100 µM resulted in the degeneration of follicles when ovarian cortical strips were cultured in vitro for 7 days (Magamage et al., 2021). Previously it was hypothesized that the inhibitory effects of KP at higher doses on cellular proliferation and viability might be due to the stimulation of apoptotic markers (Fernandois et al., 2017).

In another study conducted in our laboratory, 10 µg/ml KP favoured the expression of antiapoptotic genes (Bcl-xl, Bcl-2) but inhibited proapoptotic genes (BAX, BAD, p53 and BAK) in the cultured PFs (unpublished data). Therefore, it is possible that improvement in PF growth at 10 µg/ml in the present study might be due to the protective effect of KP against apoptosis. From among the thousands to hundreds of thousands of PFs present in the ovary at birth in mammals, almost 99% undergo apoptosis before reaching the Graafian follicle stage (Hsueh et al., 1994). Therefore KP might rescue the PFs from undergoing apoptosis.

Although there have been no published reports on the use of KP in combination with other hormones and growth factors in the culture of PFs, the present results conform with earlier reports on the in vitro development of cumulus/granulosa cells in medium supplemented with KP and other hormones (Saadeldin et al., 2012; Mamo et al., 2007; Byri et al., 2017; Fabová and Sirotkin, 2021). The positive effect of FSH in culture medium along with KP has been reported previously in sheep (Fabová and Sirotkin, 2021), in pig (Mamo et al., 2007; Saadeldin et al., 2012) and in human (Byri et al., 2017) in controlling oocyte maturation and KP activation. Recent studies have also demonstrated the role of KP in regulating the action and secretion of GH and T₄ in cultured pituitary cells (Jayasena et al., 2014). As the culture medium (SM) in the present study contained FSH, GH, IGF-1 and T₄, therefore it is assumed that KP could have a synergistic/additive effect with above growth factors and hormones for the better observed development of PFs and oocyte maturation (Table 3).

KISS1R mRNA expression in different stages of in vitro developed and in vitro cultured ovarian follicles

This study was used to elucidate the quantitative expression of kisspeptin receptor (KISS1R) in different development stages of in vivo developed follicles and their corresponding in vitro stages (see Table S1) and as evidence for the observed results in Experiments 1–3. KISS1R mRNA expression at different stages of development of in vivo developed ovarian follicles (from preantral stage onwards) in the present study alludes to (1) that KP acts through its own receptors, and (2) therefore supports the earlier view (Shahed and Young, 2009) that KP might play a direct role in follicular development. Only few reports are available to support this observation of expressing KISSR at different stages of follicular development (Liu et al., 2017; Tanyapanyachon et al., 2018). However, previous findings have demonstrated that KP receptor deficiency in mice resulted in the depletion of preantral and antral follicles, as well as the arrest of follicular development (Castellano et al., 2006). It may be argued that KISS1R gene expression need not necessarily result in the synthesis of the receptor protein, as gene transcription does not always lead to translation. However, in another study in the laboratory (unpublished data), the immunoreactivity of the KISS1R protein was observed to increase, along with the development of PFs, both in in vivo developed and cultured ovarian follicles.

A previous report observed that follicular cells (cumulus cells) were the main site for kisspeptin synthesis (Xiao et al., 2011), which explains the significantly abundant expression of KISS1R mRNA in the follicular cells observed in the present study. This finding is further supported by the earlier study that explained the basis of kisspeptin secreted in granulosa cells with regard to progesterone secretion and associated actions during steroidogenesis by regulating the steroidogenic enzymes Erk1/2 in the mitogen-activated protein kinase pathway (Ricu et al., 2012). It was also reported that KP exhibited a dose-dependent regulation of estradiol-17β secretion, in which the low doses significantly increased estradiol-17β compared with a high dose of the KP thereby suggesting the role of KP in gonadal steroid production at lower doses, which further increased the LH response to kisspeptin (Peng et al., 2013). Also, it was apparent that KISS1R expression was progressive with the formation of the antrum, which indicates the accumulation of kisspeptin in the follicular fluid, as confirmed in previous studies (Saadeldin et al., 2012; Singh et al., 2021).

Of the four groups, Group 4 showed marked increase for KISS1R mRNA expression compared with the other in vitro cultured groups. Although meagre reports are available to date to support this observation, one study showed a positive correlation between FSH and KP, which reflects the permissive effect of FSH for KP action, thereby leading to an increase in KISS1R expression (Saadeldin et al., 2012). It was also reported previously that a blockade in FSH secretion suppressed KISS1R expression (Roa et al., 2008a). Reports are available to indicate the role of LH in increasing the kisspeptin expression responsiveness to FSH (Saadeldin et al., 2012; Babitha et al., 2014; Fabová and Sirotkin, 2021). As FSH is a component of SM (Groups 2 and 4) used in this study, it is possible that FSH shows synergism with kisspeptin and also suggests a carryover effect between FSH and LH, which might be responsible for the highest expression of KISS1R in the PFs cultured in Group 4 compared with all other groups. mRNA expression of KISS1R was found to be highest in the COCs aspirated from in vivo developed large antral follicles, cumulus cells and oocytes from 6-day cultured PFs subjected to an additional 24 h of IVM subsequently during different culture conditions in this study. This observation highlights the possibility of bidirectional communication of kisspeptin in regulating the proliferation and differentiation of granulosa cells and therefore its receptor expression, as reported in previous studies (Roa et al., 2008b; Levine, 2015; Cielesh et al., 2017). Therefore, KISS1R expression in the oocytes and granulosa cells appears to regulate the preovulatory surge of gonadotrophins, which could promote oocyte survival in the later stages of follicular growth (Dorfman et al., 2014). As LH is one of the hormones supplemented to IVM medium, the high expression of KISS1R in the oocytes subjected to in vitro maturation could be attributed to the LH-dependent matrix metalloproteinase (MMP) activity, which partly supports the KISS1R expression (Shahed and Young, 2009; Liu et al., 2017) and the role of LH in the KISS1R expression ovaries as was reported previously (Roa et al., 2008a). Currently detailed quantitative investigations into the levels of gene expression of various factors related to kisspeptin in connection with ovulation and associated events of female reproduction in different stages of in vivo grown and in vitro cultured PFs in various culture media is under progress in our laboratory, and could provide a better understanding of the local role of kisspeptin in ovine reproduction.

In the present study, although the pattern of expression of KISS1R was similar among different development stages of in vivo developed and in vitro cultured ovarian follicles, quantitatively, the expression was better in the in vivo grown follicles. This suggests (1) the present culture conditions may not support normal expression, or (2) in vivo secretion of KP might be higher as observed in pig and human (Saadeldin et al., 2012; Singh et al., 2021) requiring higher concentration of homologous receptors. These facets would form interesting subjects for future studies.

In conclusion, KP has a direct influence on the growth of ovarian follicles through the stimulation of the expression of homologous receptors. It could also have a synergistic/additive effect with other growth factors and hormones in stimulating the growth of PFs under in vitro culture.