Journal of Animal Reproduction and Biotechnology 2021; 36(1): 51-58
Published online March 31, 2021
https://doi.org/10.12750/JARB.36.1.51
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Manjula Priyantha Sumith Magamage1,2 , Sriravali Sathagopam1 , Kiran Avula1 , Di Neththi Nimesh Madushanka2 and Sathya Velmurugan1,*
1National Institute of Animal Biotechnology, Hyderabad 500049, India
2Department of Livestock Production, Sabaragamuwa University, Belihuloya RN 70190, Sri Lanka
Correspondence to: Sathya Velmurugan
E-mail: sathyavet@gmail.com
ORCID https://orcid.org/0000-0003-0769-8424
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Kisspeptin, a neuropeptide and the master controller of reproductive axis upstream to GnRH neurons, and its receptor are also expressed in extrahypothalamic tissues, such as ovaries. As systemic kisspeptin has been shown to modulate follicular dynamics in cattle, we hypothesized that kisspeptin has direct actions on the ovarian follicular development. We also hypothesized that kisspeptin regulation of primordial follicle development is via modulation of VEGF expression. In order to test these hypotheses, we cultured caprine ovarian cortical strips in vitro for 7 days with supplementation of kisspeptin at 1, 10 and 100 μM concentration and observed the development of primordial follicles into intermediate, primary and secondary follicles. We also studied the alteration in the expression profile of VEGF and VEGF transcript variant 2 mRNA during follicular development in the presence of kisspeptin. We confirmed the presence of GPR54 in goat ovaries in our preliminary studies. Supplementation of kisspeptin at 1 and 10 μM concentration facilitated the development of primordial follicles into intermediate, primary and secondary follicles with less number of degenerated follicles while the same at 100 μM resulted in degeneration of follicles. We observed a drastic increase in the expression profile of VEGF and VEGF transcript variant 2 mRNA upon culture which was independent of kisspeptin treatment. In conclusion, our studies show that kisspeptin facilitates ovarian primordial development in vitro.
Keywords: GPR54, kisspeptin, ovary, primordial follicle development, VEGF
Kisspeptin, the product of the
Stringent control of repeated cycles of growth and remodeling process is required in the ovary, as oocyte must develop in an avascular environment within the follicle, in contrast to the highly vascular corpus luteum (Zimmermann et al., 2001; Reynolds et al., 2002). Hence, the mammalian ovary undergoes programmed angiogenic processes during the ovarian cycle (Stouffer et al., 2001; Reynolds et al., 2002). Only the fastest growing and most highly aggressive tumors match the high rate of vessel growth and proliferation within the ovary which is associated with a high metabolic requirement for rapid steroidogenesis (Reynolds et al., 2002). Although the process of primordial follicle recruitment is incompletely understood, specific growth factors must either stimulate primordial follicles to leave the dormant state or inhibit primordial follicles from entering the growing pool (Kim, 2012). Among many candidates, vascular endothelial growth factor-A (VEGF-A), a member of the VEGF family, has emerged as one of the most important regulator of angiogenesis in the ovary (Geva and Jaffe, 2000). The
There are limited number of studies on the role of VEGF in ovarian physiology in domestic animal species. Here we explored the expression of VEGF-A transcript variant 2 and VEGF-A165a, proangiogenic factors, during follicle development in goat ovaries
We found that kisspeptin did not suppress the increase in
Goat ovaries, along with uteri, were collected from a slaughter house and transported to the laboratory under controlled conditions. Ovaries were collected, cleaned, washed once in phosphate buffered saline (PBS) supplemented with 0.2% (w/v) cetyltrimethylammonium bromide and washed thrice in Dulbecco’s PBS (Sigma) supplemented with 0.1% (w/v) polyvinyl alcohol. Ovarian cortical strips (approximately, 2 mm × 1 mm × 0.5 mm) that contain primordial follicles were selected, and cut into two pieces (each approximately 1 mm × 1 mm × 0.5 mm); one part was fixed immediately for histological examination later to assess the follicle number and morphology and the other part was washed thrice and immersed in Leibovitz 15 medium (Sigma) before culture. The Leibovitz medium contained 0.1% (v/v) Pen-Strep as antibiotic.
In each experiment, a group of 8 cortical strips were cultured for 168 h on a floating membrane filter (diameter 25 mm and pore size 0.45 μm) in six-well culture dish with Millicell® Cell Culture Inserts (Millipore) under the humidified atmosphere of 5% CO2 and 95% air at 38℃. The basic culture medium is Dulbecco’s Minimum Essential Medium (D-MEM; Sigma) supplemented with Pen-Strep (1% v/v), sodium pyruvate (0.1 mg/mL), Insulin-Transferrin-Selenium (ITS; 0.05% v/v) and bovine serum albumin (5% v/v). During the culture period, half of the total medium was changed every 24 h. Either saline or kisspeptin-10 (1, 10 or 100 ng/mL; equivalent to 0.75, 7.59 and 75.85 nM, respectively; Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH2; synthesized from Biotech Desk) was added on day 1.
The ovarian cortical strips before and after culture were fixed in 4% (w/v) paraformaldehyde in PBS, dehydrated, embedded in paraffin wax and serially sectioned (10 μm sections), and stained with hematoxylin and eosin. Primordial follicles were identified as those having oocytes of 25-30 μm diameter containing a large spherical nucleus surrounded by small lipid droplets (Magamage et al., 2010). The numbers of different stages of follicles were recorded. The follicles were counted in every section where the oocyte nucleus was seen and hence double counting in adjacent sections was avoided. The follicles were classified into four categories according to the number and morphology of granulosa cell layers: (1) primordial follicles with a single layer of flattened granulosa cells surrounding the oocyte, (2) intermediate follicles with a single layer containing a mixture of flat and cuboidal granulosa cells, (3) primary follicles with a single layer of cuboidal granulosa cells, and (4) secondary follicles with two or more layers of cuboidal granulosa cells (Magamage et al., 2010). Degenerated follicles were identified by the staining properties of the oocyte cytoplasm and nucleus: pale cytoplasm and dark pyknotic nucleus. Furthermore, follicles having a shrunken oocyte, extensive cytoplasmic vacuolations and disintegrated granulosa cell layer were also considered to be degenerated follicles.
The ovarian cortical strips were washed thrice in PBS before and after culture; protein extraction was done using RIPA buffer and quantification using Bradford assay. Rabbit polyclonal anti-GPR54 antibody (GeneTex; Cat No. GTX37417) was used for probing. HRP-conjugated goat anti-rabbit IgG antibody was used as the secondary antibody. A Western blotting detection kit (ECL Prime; GE; Cat. No. RPN2232) was used for detection.
The ovarian cortical strips were washed thrice in PBS before and after culture and RNA extraction was done using RNEasy plus mini kit (Qiagen) following the manufacturer’s protocol and quantified by Nano Drop 2000 (Thermo Scientific). cDNA was synthesized using a kit (Takara; Cat # 6110A) by reverse transcription using 200 ng of RNA. The sequences of the primer sets (IDT) are given in Table 1. The assays were performed in a Real Time PCR System (Applied Biosystems 7500). β-actin was used to normalize the input of cDNA in qPCR. The ratio of target gene compared with β-actin amplification represented the relative levels in each sample. Results were calculated based on Ct values using the formula 2∧-[Ct (gene)-Ct (β-actin)] as described in the user manual (Applied Biosystems).
Table 1 . Primers used for qPCR
VEGF-A165a | |
Forward primer | GTTCAGAGCGGAGAAAGCAT |
Reverse primer | TCACATCTGCAAGTACGTTCG |
Capra hircus VEGF-A transcript variant 2 | |
Forward primer | AACCTGACATGAAGGAAGAGGGAG |
Reverse primer | CGGTGATTTAGCAGCAAGAGAA |
Capra hircus Kisspeptin 1 | |
Forward primer | TCCCTCCCTTCTTTCCTTCCTAA |
Reverse primer | AGGGACGAGCCTGAACCGA |
Capra hircus GPR54 | |
Forward primer | GTCTGGGAAGAAGGTTGGGAG |
Reverse primer | CCGTCTTGGGTTTCCATTGTG |
Capra hircus β-actin | |
Forward primer | GTCACCAACTGGGACGACAT |
Reverse primer | CATCTTCTCACGGTTGGCCT |
Data were compiled using Excel (Microsoft Inc). GraphPad Prism (Version 6.07, GraphPad Software Inc.) statistical software was used to analyse the data using one-way ANOVA followed by Tukey’s multiple comparison tests. Differences were considered to be significant when
In preliminary studies, the presence of kisspeptin receptor, GPR54, in ovarian cortical strips, was confirmed by Western blot analysis (Fig. 1).
Kisspeptin treatment induced development of primordial follicles into primary and secondary follicles upon culture. Representative histological sections depicting various stages of follicle development with respect to treatments are shown in Fig. 2.
On day 0, prior to culture, the cortical strips predominantly contained primordial follicles. Upon culture without any treatment, there was no development of these follicles. Upon supplementation of kisspeptin, primordial follicles developed into intermediate, primary and secondary follicles. While kisspeptin at 1 ng/mL concentration facilitated the development of primordial follicles into intermediate, primary and secondary follicles with least number of degenerated follicles, 10 ng/mL kisspeptin facilitated the same with lesser number of degenerated follicles (Fig. 3A to 3E). Degenerated follicles were the highest with kisspeptin at 100 ng/mL concentration (Fig. 3E). Total number of viable follicles were the highest in 1 ng/mL kisspeptin-treated strips and the least in 100 ng/mL kisspeptin-treated strips (Fig. 3F).
There was no significant change in the expression of
Kisspeptin, acting via its receptor, is a critical regulator of the reproductive axis by stimulating hypothalamic GnRH release (Caraty and Franceschini, 2008). More recently, studies have suggested that kisspeptin may also have direct gonadal effects due to the presence of GPR54 in ovaries (Castellano et al., 2006). The current study on the role of kisspeptin on VEGF expression and on primordial follicle development in mammalian ovaries delineates yet another significant role played by kisspeptin in ovaries.
The majority of the follicles in the ovary are the primordial follicles (Findlay et al., 2015). Though sporadic development of these follicles into other developmental stages are observed even before puberty, development of these follicles into advanced stages, such as tertiary and ovulatory follicles, occur upon the influence of reproductive hormones that are released during regular oestrous or menstrual cycles. Facilitation of development of primordial follicles into other subsequent stages, by kisspeptin, as observed in our study, demonstrates that hypothalamic or ovarian kisspeptin plays an important role in the regulation of ovarian physiology, as reviewed recently (Clarke et al., 2015).
Angiogenesis is an important event during primordial follicle development as the blood supply increases to support the growing follicle (Fraser, 2006). Hence, increase in the expression of VEGF during follicle development may be expected. However, increase in the same in the 7-day cultured control strips, in which there is no significant follicle development was observed, is intriguing.
Kisspeptin, while inhibiting angiogenesis during metastasis, did not inhibit VEGF, an angiogenic factor, in the ovarian strips, as observed in our study. Though most of the studies have emphasized the anti-angiogenic effect of kisspeptin, especially during tumour growth, a few studies have shown that kisspeptin increases migration and proliferation of endothelial cells, the components of angiogenesis (Golzar and Javanmard, 2015). Facilitation by kisspeptin of ovarian follicular growth has been observed
In conclusion, this study demonstrates a direct action of kisspeptin on ovarian cells facilitating primordial follicle development. In addition, lack of inhibition of VEGF expression by kisspeptin suggests proangiogenic effects of kisspeptin in the ovary. The results from this
Research infrastructure and manpower support by NIAB to SV is acknowledged.
The study was conducted at NIAB. Authors are not affiliated with NIAB at the time of publication.
Funding was partially supported by India Science and Research Fellowship to MPSM by the Department and Ministry of Science and Technology, Government of India. KA received Junior Research Fellowship from a project (No. PR12395) funded by Department of Biotechnology, Government of India.
MPSM conceived the idea, designed and performed experiments, compiled and analyzed the data; SS assisted in culture experiments, performed qPCR and Western blot, and assisted in microtome sectioning; KA collected samples, and assisted in culture experiments; DNNM assisted in data analysis; SV compiled and analyzed the data, prepared the figures and wrote the manuscript.
Journal of Animal Reproduction and Biotechnology 2021; 36(1): 51-58
Published online March 31, 2021 https://doi.org/10.12750/JARB.36.1.51
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Manjula Priyantha Sumith Magamage1,2 , Sriravali Sathagopam1 , Kiran Avula1 , Di Neththi Nimesh Madushanka2 and Sathya Velmurugan1,*
1National Institute of Animal Biotechnology, Hyderabad 500049, India
2Department of Livestock Production, Sabaragamuwa University, Belihuloya RN 70190, Sri Lanka
Correspondence to:Sathya Velmurugan
E-mail: sathyavet@gmail.com
ORCID https://orcid.org/0000-0003-0769-8424
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Kisspeptin, a neuropeptide and the master controller of reproductive axis upstream to GnRH neurons, and its receptor are also expressed in extrahypothalamic tissues, such as ovaries. As systemic kisspeptin has been shown to modulate follicular dynamics in cattle, we hypothesized that kisspeptin has direct actions on the ovarian follicular development. We also hypothesized that kisspeptin regulation of primordial follicle development is via modulation of VEGF expression. In order to test these hypotheses, we cultured caprine ovarian cortical strips in vitro for 7 days with supplementation of kisspeptin at 1, 10 and 100 μM concentration and observed the development of primordial follicles into intermediate, primary and secondary follicles. We also studied the alteration in the expression profile of VEGF and VEGF transcript variant 2 mRNA during follicular development in the presence of kisspeptin. We confirmed the presence of GPR54 in goat ovaries in our preliminary studies. Supplementation of kisspeptin at 1 and 10 μM concentration facilitated the development of primordial follicles into intermediate, primary and secondary follicles with less number of degenerated follicles while the same at 100 μM resulted in degeneration of follicles. We observed a drastic increase in the expression profile of VEGF and VEGF transcript variant 2 mRNA upon culture which was independent of kisspeptin treatment. In conclusion, our studies show that kisspeptin facilitates ovarian primordial development in vitro.
Keywords: GPR54, kisspeptin, ovary, primordial follicle development, VEGF
Kisspeptin, the product of the
Stringent control of repeated cycles of growth and remodeling process is required in the ovary, as oocyte must develop in an avascular environment within the follicle, in contrast to the highly vascular corpus luteum (Zimmermann et al., 2001; Reynolds et al., 2002). Hence, the mammalian ovary undergoes programmed angiogenic processes during the ovarian cycle (Stouffer et al., 2001; Reynolds et al., 2002). Only the fastest growing and most highly aggressive tumors match the high rate of vessel growth and proliferation within the ovary which is associated with a high metabolic requirement for rapid steroidogenesis (Reynolds et al., 2002). Although the process of primordial follicle recruitment is incompletely understood, specific growth factors must either stimulate primordial follicles to leave the dormant state or inhibit primordial follicles from entering the growing pool (Kim, 2012). Among many candidates, vascular endothelial growth factor-A (VEGF-A), a member of the VEGF family, has emerged as one of the most important regulator of angiogenesis in the ovary (Geva and Jaffe, 2000). The
There are limited number of studies on the role of VEGF in ovarian physiology in domestic animal species. Here we explored the expression of VEGF-A transcript variant 2 and VEGF-A165a, proangiogenic factors, during follicle development in goat ovaries
We found that kisspeptin did not suppress the increase in
Goat ovaries, along with uteri, were collected from a slaughter house and transported to the laboratory under controlled conditions. Ovaries were collected, cleaned, washed once in phosphate buffered saline (PBS) supplemented with 0.2% (w/v) cetyltrimethylammonium bromide and washed thrice in Dulbecco’s PBS (Sigma) supplemented with 0.1% (w/v) polyvinyl alcohol. Ovarian cortical strips (approximately, 2 mm × 1 mm × 0.5 mm) that contain primordial follicles were selected, and cut into two pieces (each approximately 1 mm × 1 mm × 0.5 mm); one part was fixed immediately for histological examination later to assess the follicle number and morphology and the other part was washed thrice and immersed in Leibovitz 15 medium (Sigma) before culture. The Leibovitz medium contained 0.1% (v/v) Pen-Strep as antibiotic.
In each experiment, a group of 8 cortical strips were cultured for 168 h on a floating membrane filter (diameter 25 mm and pore size 0.45 μm) in six-well culture dish with Millicell® Cell Culture Inserts (Millipore) under the humidified atmosphere of 5% CO2 and 95% air at 38℃. The basic culture medium is Dulbecco’s Minimum Essential Medium (D-MEM; Sigma) supplemented with Pen-Strep (1% v/v), sodium pyruvate (0.1 mg/mL), Insulin-Transferrin-Selenium (ITS; 0.05% v/v) and bovine serum albumin (5% v/v). During the culture period, half of the total medium was changed every 24 h. Either saline or kisspeptin-10 (1, 10 or 100 ng/mL; equivalent to 0.75, 7.59 and 75.85 nM, respectively; Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH2; synthesized from Biotech Desk) was added on day 1.
The ovarian cortical strips before and after culture were fixed in 4% (w/v) paraformaldehyde in PBS, dehydrated, embedded in paraffin wax and serially sectioned (10 μm sections), and stained with hematoxylin and eosin. Primordial follicles were identified as those having oocytes of 25-30 μm diameter containing a large spherical nucleus surrounded by small lipid droplets (Magamage et al., 2010). The numbers of different stages of follicles were recorded. The follicles were counted in every section where the oocyte nucleus was seen and hence double counting in adjacent sections was avoided. The follicles were classified into four categories according to the number and morphology of granulosa cell layers: (1) primordial follicles with a single layer of flattened granulosa cells surrounding the oocyte, (2) intermediate follicles with a single layer containing a mixture of flat and cuboidal granulosa cells, (3) primary follicles with a single layer of cuboidal granulosa cells, and (4) secondary follicles with two or more layers of cuboidal granulosa cells (Magamage et al., 2010). Degenerated follicles were identified by the staining properties of the oocyte cytoplasm and nucleus: pale cytoplasm and dark pyknotic nucleus. Furthermore, follicles having a shrunken oocyte, extensive cytoplasmic vacuolations and disintegrated granulosa cell layer were also considered to be degenerated follicles.
The ovarian cortical strips were washed thrice in PBS before and after culture; protein extraction was done using RIPA buffer and quantification using Bradford assay. Rabbit polyclonal anti-GPR54 antibody (GeneTex; Cat No. GTX37417) was used for probing. HRP-conjugated goat anti-rabbit IgG antibody was used as the secondary antibody. A Western blotting detection kit (ECL Prime; GE; Cat. No. RPN2232) was used for detection.
The ovarian cortical strips were washed thrice in PBS before and after culture and RNA extraction was done using RNEasy plus mini kit (Qiagen) following the manufacturer’s protocol and quantified by Nano Drop 2000 (Thermo Scientific). cDNA was synthesized using a kit (Takara; Cat # 6110A) by reverse transcription using 200 ng of RNA. The sequences of the primer sets (IDT) are given in Table 1. The assays were performed in a Real Time PCR System (Applied Biosystems 7500). β-actin was used to normalize the input of cDNA in qPCR. The ratio of target gene compared with β-actin amplification represented the relative levels in each sample. Results were calculated based on Ct values using the formula 2∧-[Ct (gene)-Ct (β-actin)] as described in the user manual (Applied Biosystems).
Table 1. Primers used for qPCR.
VEGF-A165a | |
Forward primer | GTTCAGAGCGGAGAAAGCAT |
Reverse primer | TCACATCTGCAAGTACGTTCG |
Capra hircus VEGF-A transcript variant 2 | |
Forward primer | AACCTGACATGAAGGAAGAGGGAG |
Reverse primer | CGGTGATTTAGCAGCAAGAGAA |
Capra hircus Kisspeptin 1 | |
Forward primer | TCCCTCCCTTCTTTCCTTCCTAA |
Reverse primer | AGGGACGAGCCTGAACCGA |
Capra hircus GPR54 | |
Forward primer | GTCTGGGAAGAAGGTTGGGAG |
Reverse primer | CCGTCTTGGGTTTCCATTGTG |
Capra hircus β-actin | |
Forward primer | GTCACCAACTGGGACGACAT |
Reverse primer | CATCTTCTCACGGTTGGCCT |
Data were compiled using Excel (Microsoft Inc). GraphPad Prism (Version 6.07, GraphPad Software Inc.) statistical software was used to analyse the data using one-way ANOVA followed by Tukey’s multiple comparison tests. Differences were considered to be significant when
In preliminary studies, the presence of kisspeptin receptor, GPR54, in ovarian cortical strips, was confirmed by Western blot analysis (Fig. 1).
Kisspeptin treatment induced development of primordial follicles into primary and secondary follicles upon culture. Representative histological sections depicting various stages of follicle development with respect to treatments are shown in Fig. 2.
On day 0, prior to culture, the cortical strips predominantly contained primordial follicles. Upon culture without any treatment, there was no development of these follicles. Upon supplementation of kisspeptin, primordial follicles developed into intermediate, primary and secondary follicles. While kisspeptin at 1 ng/mL concentration facilitated the development of primordial follicles into intermediate, primary and secondary follicles with least number of degenerated follicles, 10 ng/mL kisspeptin facilitated the same with lesser number of degenerated follicles (Fig. 3A to 3E). Degenerated follicles were the highest with kisspeptin at 100 ng/mL concentration (Fig. 3E). Total number of viable follicles were the highest in 1 ng/mL kisspeptin-treated strips and the least in 100 ng/mL kisspeptin-treated strips (Fig. 3F).
There was no significant change in the expression of
Kisspeptin, acting via its receptor, is a critical regulator of the reproductive axis by stimulating hypothalamic GnRH release (Caraty and Franceschini, 2008). More recently, studies have suggested that kisspeptin may also have direct gonadal effects due to the presence of GPR54 in ovaries (Castellano et al., 2006). The current study on the role of kisspeptin on VEGF expression and on primordial follicle development in mammalian ovaries delineates yet another significant role played by kisspeptin in ovaries.
The majority of the follicles in the ovary are the primordial follicles (Findlay et al., 2015). Though sporadic development of these follicles into other developmental stages are observed even before puberty, development of these follicles into advanced stages, such as tertiary and ovulatory follicles, occur upon the influence of reproductive hormones that are released during regular oestrous or menstrual cycles. Facilitation of development of primordial follicles into other subsequent stages, by kisspeptin, as observed in our study, demonstrates that hypothalamic or ovarian kisspeptin plays an important role in the regulation of ovarian physiology, as reviewed recently (Clarke et al., 2015).
Angiogenesis is an important event during primordial follicle development as the blood supply increases to support the growing follicle (Fraser, 2006). Hence, increase in the expression of VEGF during follicle development may be expected. However, increase in the same in the 7-day cultured control strips, in which there is no significant follicle development was observed, is intriguing.
Kisspeptin, while inhibiting angiogenesis during metastasis, did not inhibit VEGF, an angiogenic factor, in the ovarian strips, as observed in our study. Though most of the studies have emphasized the anti-angiogenic effect of kisspeptin, especially during tumour growth, a few studies have shown that kisspeptin increases migration and proliferation of endothelial cells, the components of angiogenesis (Golzar and Javanmard, 2015). Facilitation by kisspeptin of ovarian follicular growth has been observed
In conclusion, this study demonstrates a direct action of kisspeptin on ovarian cells facilitating primordial follicle development. In addition, lack of inhibition of VEGF expression by kisspeptin suggests proangiogenic effects of kisspeptin in the ovary. The results from this
Research infrastructure and manpower support by NIAB to SV is acknowledged.
The study was conducted at NIAB. Authors are not affiliated with NIAB at the time of publication.
Funding was partially supported by India Science and Research Fellowship to MPSM by the Department and Ministry of Science and Technology, Government of India. KA received Junior Research Fellowship from a project (No. PR12395) funded by Department of Biotechnology, Government of India.
MPSM conceived the idea, designed and performed experiments, compiled and analyzed the data; SS assisted in culture experiments, performed qPCR and Western blot, and assisted in microtome sectioning; KA collected samples, and assisted in culture experiments; DNNM assisted in data analysis; SV compiled and analyzed the data, prepared the figures and wrote the manuscript.
Table 1 . Primers used for qPCR.
VEGF-A165a | |
Forward primer | GTTCAGAGCGGAGAAAGCAT |
Reverse primer | TCACATCTGCAAGTACGTTCG |
Capra hircus VEGF-A transcript variant 2 | |
Forward primer | AACCTGACATGAAGGAAGAGGGAG |
Reverse primer | CGGTGATTTAGCAGCAAGAGAA |
Capra hircus Kisspeptin 1 | |
Forward primer | TCCCTCCCTTCTTTCCTTCCTAA |
Reverse primer | AGGGACGAGCCTGAACCGA |
Capra hircus GPR54 | |
Forward primer | GTCTGGGAAGAAGGTTGGGAG |
Reverse primer | CCGTCTTGGGTTTCCATTGTG |
Capra hircus β-actin | |
Forward primer | GTCACCAACTGGGACGACAT |
Reverse primer | CATCTTCTCACGGTTGGCCT |
print Article | |
Export to Citation | Open Access |
Google Scholar | Send to Email |
pISSN: 2671-4639
eISSN: 2671-4663