Effects of quinestrol treatment on regional protein expression in the rat testis and epididymis: Insights from SDS-Page, Western Blotting, and Elisa

Introduction

The world's population has grown by 380 million people since the year 2000 to reach 6.5 billion. Despite reduced birth rates, the world population is estimated to reach 9.7 billion by the year 2050 and continue growing at a rate of 34 million people per year by the middle of the century, with the vast majority of these people living in developing nations (Leridon, 2020) [21] . The huge number of unwanted, usually adolescent pregnancies is another major issue. Over the last 50 years, there has been a steady improvement in contraceptive research. Despite considerable advancements in contraception alternatives for women over the last 50 years, the global population continues to rise at an alarming rate. Large-scale programs to meet the world's unmet family planning requirements have collected and driven scientists, healthcare providers, program managers, and users to re-examine the many techniques of contraception, focusing on those that have been demonstrated to be more effective (Sitruk-Ware et al., 2013) [28] . Birth control is an essential means to regulate the population and control fertility rates so that it does not hinder the socio-economic growth of the nation. Contraception is the deliberate prevention of pregnancy using various methods, including behavioral, pharmaceutical, and surgical techniques. Effective contraception enables couples to engage in sexual activity without the risk of unintended pregnancy while providing flexibility for family planning. Modern contraceptives influence estrogen and progesterone levels and include oral contraceptive pills, long-acting injections, implants, transdermal patches, and intrauterine devices (Hubacher & Trussell, 2015) [15] . Fertility regulation is categorized into hormonal and non-hormonal contraception. Hormonal contraceptives contain synthetic estrogen, such as ethinyl estradiol, or synthetic progestin, primarily in oral form but also available as implants and vaginal inserts (Colquitt & Martin, 2017) [6] . A common combination, quinestrol and levonorgestrel in a 1:2 ratio, offers high efficacy with minimal side effects (Wright et al., 2020) [34] . Progestin prevents ovulation by inhibiting the luteinizing hormone surge, thickening cervical mucus, reducing tubal motility, and thinning the endometrial lining. Estrogen enhances effectiveness by suppressing follicle-stimulating hormone and potentiating progestin's action on the luteinizing hormone surge (Kiley & Hammond, 2007; Dhont, 2010) [19,9] . Quinestrol, a long-lasting, fat-soluble estrogen derivative of ethinyl estradiol, has been used in recent years to limit fertility by altering the structure and function of reproductive organs. It is a major long-term contraceptive taken by women for extended periods. Levonorgestrel and quinestrol, combined in a 2:1 ratio, are commercially used as female contraceptive medicines. Similar to its contraceptive efficacy in females, various steroid hormone combinations have been explored for male contraception, demonstrating potential antifertility effects. After gastrointestinal absorption, quinestrol is stored in adipose tissue and released gradually, contributing to its long-lasting impact. As a stable estradiol homolog, it suppresses the hypothalamic-pituitary-gonadal axis, reducing gonadotropin-releasing hormone secretion and impairing fertility regulation (James et al., 2020) [17] . The hypothalamic-pituitary-gonadal axis is the primary endocrine feedback mechanism controlling reproductive function. Its suppression leads to decreased levels of follicle-stimulating hormone, luteinizing hormone, and testosterone or estradiol, which disrupts spermatogenesis and ovulation. While androgens and progesterone can reduce male fertility, high doses of estrogen and progesterone effectively inhibit female ovulation, suppressing follicle-stimulating hormone and luteinizing hormone production and preventing luteinizing hormone surges. Studies indicate that estrogen treatment has a stronger impact on male infertility compared to androgens, as it directly suppresses the hypothalamic-pituitary-gonadal axis, reduces testosterone levels, and obstructs normal spermatogenesis, ultimately leading to male infertility (Dacheux & Dacheux, 2014) [8] . Xenoestrogens, which mimic estrogen, further reduce male fertility by disrupting Leydig cell function, either directly or through indirect effects on the hypothalamic-pituitary-gonadal axis (Sullivan & Mieusset, 2016) [29] . Research suggests that estrogen could be an effective agent for fertility control in male rats. Xenoestrogens have been linked to testicular hypoplasia, rete testis tumors, and erectile dysfunction. Quinestrol, apart from being a human contraceptive, is also used as a rodenticide to control wild rat populations. High doses of quinestrol have been shown to induce antifertility effects in laboratory rodents. Various xenoestrogens, including diethylstilbestrol, estradiol-17, and phytoestrogens, have been reported to reduce testosterone production in rodents, birds, and mammals, leading to decreased fertility. Early studies on orally active estrogens, progestagens, and androgens demonstrated significant disruptions in ovulation, implantation, and spermatogenesis. Quinestrol alone or in combination alters reproductive organ structures, reducing male fertility in rats and wild rodents. Several laboratory and field studies confirm that the compound EP-1 effectively decreases fertility in multiple wild rodent species (Chakraborty & Saha, 2022) [3] . The antifertility effects of quinestrol are primarily due to oxidative stress, germ cell apoptosis, and reduced plasma testosterone levels. Any disturbance in the hypothalamicpituitary-testicular axis, which regulates sexual differentiation, development, and fertility, can lead to male gonadal dysfunction. Spermatogenesis commences in the seminiferous tubules of the testis and begins at the basal membrane and continues to the tubule lumen via the outermost portion of the Sertoli cells that border the seminiferous tubules (Reisse et al., 2001) [25] . It is a continuous process in males by which mature and functionally active spermatozoa are produced in male gonads. Spermatozoa that leave the testis and enter the epididymis, a long convoluted tubule, are non-functional gametes (Gervasi & Visconti, 2016) [11] . The epididymis is a crescent-shaped, convoluted part of the male reproductive system, a long tube that links male gonads to the male duct, the vas deferens, and it is derived from the Wolffian duct and is mostly made up of mesenchymal tissue during birth (Turner, 2008) [31] . It is partitioned into three primary segments: caput, corpus, and cauda, each of which is further divided into intra-segmental zones delimited by connective tissue septa and expresses a distinct pattern of genes (Sullivan & Mieusset, 2016) [29] .

Understanding the qualitative and quantitative changes in tissue homogenate of testis and epididymis, epididymal fluid and sperm membrane extract (SME) is crucial for elucidating the molecular mechanisms underlying quinestrol-induced infertility. Therefore, this study aims to determine the molecular changes in spermatozoa during the maturation process in response to quinestrol exposure using advanced biochemical techniques such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and enzyme-linked immunosorbent assay (ELISA).

Materials and Methods

The procurement, acclimatization, and maintenance of male albino rats were conducted in strict compliance with ethical and regulatory standards to ensure the welfare of the animals and the reliability of the study. Proper housing conditions, controlled environmental factors, and a balanced diet were maintained to minimize stress and variability. Institutional approvals and adherence to CPCSEA guidelines reinforced the ethical integrity of the research. By following these protocols, the study upheld scientific rigor while ensuring humane treatment, contributing to the validity and reproducibility of the experimental outcomes.

Drug used for the experiment

To evaluate the effects of quinestrol, synthetic estradiol (17 alpha-Ethinylestradiol 3-cyclopentltl ether, C 2 H 32 O 2, 364 g/mol) a commercial formulation was acquired from CDH Chemicals and administered to the rats by oral intubation.

Dose used in the experiment

Rats were acclimated to lab conditions for a period of fifteen days prior to the experiment. All 40 male rats were separated into two groups (n=20) after acclimatization.

Group 1: Control Group 2: 2mg/kg quinestrol body weight Every dosage that contains quinestrol dissolves in Dimethyl sulphoxide because quinestrol dissolves in an organic solvent. For ten days, oral intubation was used as the primary mode of treatment.

Autopsy of rats

After the course of treatment was completed, the rats were dissected. The testicles and epididymides were taken out and their adhering tissue was removed in order to weighed them precisely.

Extraction of Caput, Corpus, and Cauda Sperm Membrane Proteins

The membrane proteins of caput, corpus, and cauda sperm were extracted using SDS as described by Jones et al.

(1983) [35] . After two washes with PBS (pH 7.4), spermatozoa (13 × 10⁶) were suspended in 1.0 ml of 15 mM DOC/2% SDS in 62.5 mM Tris-HCl (pH 8.0), containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 25 mM benzidine, and 10 mM aprotinin). The sperm suspensions were sonicated three times at 20 watts for 20 seconds, followed by centrifugation at 10,000 g for 30 minutes to collect the supernatant. This process was repeated twice, first with centrifugation at 20,000 g for 30 minutes and then at 10,000 g for 45 minutes. The SMEs were collected from the pellets and stored in 0.1 ml aliquots at -20°C until needed.

Preparation of testis and epididymal tissue homogenates for SDS-PAGE and fluid collection from caput, corpus, and cauda regions of the epididymis.

The testis and epididymal tissues, including the caput, corpus, and cauda regions, were homogenized by combining the tissue with a known quantity of 0.1 M phosphatebuffered saline (pH 7.6). The homogenization process was carried out using a tissue homogenizer. The entire homogenate was then centrifuged in a chilled microcentrifuge at 3000 revolutions per minute (825 relative centrifugal force) for 10 minutes. The tissue homogenates of the testis and epididymis was collected for further studies.

Similarly fluid was collected from three regions of the epididymis i.e caput, corpus and cauda for further studies.

Protein Extraction from Animal Tissues (Cilia et al.,

2009) [5] Trichloroacetic acid (TCA)-acetone method was used for the extraction of proteins from testis and epididymis tissues.

Frozen tissue was homogenized in ice-chilled PBS, and the homogenate was then mixed with 10% TCA in 10 ml acetone containing 2% beta-mercaptoethanol. The mixture was stirred by inverting the tube ten times and incubated at -20°C for at least 12 hours to facilitate protein precipitation. The precipitated protein was centrifuged at 5000 g for 30 minutes. The resulting pellet was washed ten times with 10 ml of ice-cold acetone, with vigorous disruption using a crusher between washes. The protein was then air-dried and stored at -20°C until further use.

Sample Preparation for SDS-PAGE using TCA Precipitation

The extract was mixed with TCA at a final concentration of 10-20% and incubated on ice for 30 minutes for protein precipitation. The sample was then centrifuged at 14,000 rpm for 30 minutes at 4°C in a microcentrifuge. The pellet was then washed by centrifuging again at 14,000 rpm for 10 minutes, followed by draining the acetone. The washing step was repeated, and any residual acetone was removed by air drying. The dried sample was resuspended in 20-30 µl of sample buffer containing β-mercaptoethanol, heated at 95°C for 1-2 minutes, and briefly spun in a table centrifuge. The sample was then ready for SDS-PAGE analysis.

Protein qualitative analysis using SDS-PAGE (Laemmli, 1970) [20] SDS-PAGE was performed following the method described by Laemmli (1970) [20] . Resolving and stacking gels were prepared using electrophoresis apparatus. Protein samples (20 µg) were mixed with sample buffer and heated in boiling water for 5-10 minutes before being loaded into the wells of the gel. The electrophoresis apparatus was run, and the gel was stained with 0.5% Coomassie Brilliant Blue R-250 solution for 20-30 minutes. The gel was then destained using a solution containing 20% methanol, 10% glacial acetic acid, and 70% distilled water, with multiple changes of the destaining solution until distinct protein bands were visible. The molecular weight of protein bands in different lanes was recorded in kilodaltons (kDa) after comparison with a protein marker.

Detection of sperm membrane proteins by western blotting (Towbin et al., 1979) [30] Membrane proteins from caput, corpus, and cauda-SME were identified using western blotting. Proteins from cauda-SME were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (NCM) using a blot sandwich setup in transfer buffer. Transfer was performed at 100 V for 2-3 hours with cooling pads to prevent overheating.

Transfer quality was assessed with Ponceau Dye, followed by washing. The membrane was blocked overnight at 4°C, washed with phosphate buffer saline (PBS), and incubated with the primary antibody for 2-3 hours at 37°C. After washing, it was incubated with HRPO-conjugated anti-rat IgG (1:10,000) for 2-3 hours, followed by further washing. Detection was performed using a substrate solution containing DAB, 4-chloronaphthol, and H₂O₂ for 10 minutes. The reaction was stopped by washing with distilled water.

Detection/Titration of Anti-Cauda SDS-SME Antibodies by ELISA

LDH-C like proteins in tissue and SMEs from the epididymis were quantified using indirect ELISA (Crowther, 1995) [7] . A 96-well ELISA plate was coated with 100 µl of antigen (2 µg/100 µl) and incubated at 37°C for 3 hours, followed by PBS washing. Blocking was done with 2% BSA for 2 hours at 37°C, then washed again. Anti-LDH-C antibody was added and incubated overnight at 4°C, followed by PBS washing. HRPO-conjugated anti-rat IgG (1:10,000) was added and incubated for 3 hours at 37°C, followed by washing. The reaction was developed with OPD-H₂O₂ substrate for 20 minutes in the dark, stopped with 5N H₂SO₄, and absorbance was measured at 492 nm using an ELISA reader. This revealed that treatment with quinestrol affects processes of protein synthesis in male albino rats. Also, oxidative stress can raise free radical levels in cells, which leads to protein oxidation. Proteins oxidise when their molecules interact with the radicals. This causes the protein's structure to alter and become aberrant, further this indicates inhibition of transcription and translation, eventually resulting in loss of some proteins (Hayati et al. 2010) [16] .

Statistical Analysis

Qualitative analysis of proteins by SDS-PAGE of epididymal fluid of three regions of epididymis

The SDS-PAGE analysis of epididymal fluid from control and quinestrol-treated rats revealed significant changes in protein profiles across the caput, corpus, and cauda regions of the epididymis. The trend of height, raw volume and % raw volume in protein files between control and treated rats were presented in table 6-8 and figure 2. In control rats, proteins with molecular weights of 100, 80, 55, 40, 20, and 14 kDa were observed, whereas treated rats exhibited proteins of 80, 55, 40, 35, 30, and 25 kDa. Notably, proteins of 20 and 14 kDa were absent in treated rats, while new bands corresponding to 30, 25, and 35 kDa appeared in the epididymal fluid of treated rats. This suggests that quinestrol administration alters the protein composition of the epididymal fluid, potentially due to oxidative stress-induced damage or disruptions in the epididymal microenvironment, which is critical for sperm maturation (Shen et al., 2011; Jones, 1998) [26,18] .

Qualitative analysis of proteins by SDS-SMEs of three regions of epididymis

The SDS-PAGE indicated the presence 6 proteins corresponding to molecular weight of 70, 57, 40, 36, 35, 25 kDa in the SME of caput, corpus and cauda epididymal fluid of control rats and similarly 70, 57, 40, 36, 35, 25 kDa proteins were observed in the group treated with quinestrol. The trend of height, raw volume and % raw volume in protein files between control and treated rats were presented in table 9-11 and figure 3. In the present study it has been reported that quality and quantity of sperm membrane proteins have been decreased after quinestrol treatment.

Detection of Lactate dehydrogenase-C proteins by western blotting

The SDS-PAGE indicated the presence of 70, 57, 40, 36, 35, 25 kDa proteins in the SME of caput, corpus and cauda of control rats and similarly 70, 57, 40, 36, 35, 25 kDa proteins were observed in the group treated with quinestrol @ 2 mg/Kg body weight for a period of 10 days. The results of SDS-PAGE of SME of three regions of indicated that protein quantity decreases in comparison to control group. The 35 kDa protein band was observed in tissue homogenate of testis and epididymis and in SME of control and treated rats but this band was absent in epididymal fluid of control and treated rats. The presence of this protein in testis and SME indicated that after secreting from tissue it might get absorbed on the surface of sperm in the epididymis. In SME of three regions of epididymis a 35 kDa protein was identified which had remarkable role in male fertility. The results on the NCM membrane (figure 4) showed that the quantity of the targeted protein i.e. LDH-C after reaction with selected antibody in the caput, corpus and SME decreases after quinestrol as compared to the control group.

Determination of LDH-C concentration by ELISA

Western blotting results indicated the presence of 35 kDa protein in caput, corpus and cauda SME of control and treated rats. In the present study, the characteristic expression of particular protein i.e LDH-C was analysed in extract of testis and SME of caput, corpus and cauda using ELISA. The results are depicted in table 12 and figure 5. Quantity of proteins was estimated from the absorbance at 492 nm and was calculated by determining the per cent change.

The per cent age change in testis of control and treated rats was 0.73±0.12 and 0.55±0.09. Similarly, per cent age change in caput, corpus and cauda of control and treated rats was 0.21±0.09, 0.18±0.05, 0.68±0.14 and 0.15±0.07, 0.11±0.01, 0.41±0.08. The data showed that there was significant difference between the control and treated rats. This revealed that quantity of LDH-C decreases after quinestrol treatment in male albino rats. Similar trend was observed in quantity of caput, corpus and cauda which indicated the quantity in the treated rats decreases after quinestrol treatment.

Discussion

The present study highlights the profound effects of quinestrol on protein expression in the male reproductive system, particularly in the testis and epididymis. The SDS-PAGE analysis revealed significant alterations in protein band patterns between control and quinestrol-treated rats, indicating that quinestrol affects protein synthesis, degradation, and expression, ultimately disrupting critical processes involved in spermatogenesis, sperm maturation, and fertilization.

The findings demonstrate that while certain testicular proteins (70, 67, 45, 35, 24, and 17 kDa) remained consistently present in both control and treated rats, epididymal protein expression was significantly altered by quinestrol. Notably, the 55 kDa protein band was absent in the caput and corpus epididymis of treated rats, suggesting disruption in sperm maturation. Additionally, the loss of the 15 kDa band in the cauda epididymis highlights quinestrol's potential adverse effects on sperm function.

A critical observation was the reduced intensity and raw volume of the 67 kDa protein band, corresponding to estrogen receptor-α (ERα). ERα is crucial for testicular function and sperm viability, and its downregulation in quinestrol-treated rats may contribute to impaired spermatogenesis and steroidogenesis (Hess et al., 1997; Cavaco et al., 2009) [14,2] . Similarly, decreased expression of the 45 kDa band, associated with acrosomal protein SP-10, suggests a potential impairment in sperm-egg interaction and fertilization (Naz & Vanek, 1998; Batista et al., 2022) [23,1] . The absence of the 55 kDa band, corresponding to spermassociated fertilin-β, in the caput and corpus further supports the hypothesis that quinestrol impairs sperm-egg fusion (Cho et al., 1998; Gundogan et al., 2022) [4,13] .

The epididymal fluid analysis revealed substantial protein modifications, with the appearance of new proteins (30 and 25 kDa in the caput, 30 kDa in the corpus, and 35 and 25 kDa in the cauda) in treated rats. Conversely, the loss of 20 and 14 kDa proteins across all epididymal regions suggests that quinestrol disrupts the normal protein acquisition process essential for sperm maturation. These changes are consistent with oxidative stress-induced protein oxidation, which damages reproductive organ membranes and interferes with sperm-epididymal interactions (Shen et al., 2011) [26] . Epididymal secretory proteins are essential for sperm function, with previous studies highlighting their role in sperm motility and fertilization (Veron et al., 1982; Gonzalez et al., 1984; Jones, 1998) [32,12,18] . Quinestrolinduced alterations in the epididymal microenvironment may result in significant disruptions to these processes, negatively impacting fertility. The decreased presence of key sperm membrane proteins, particularly those involved in sperm-egg binding, further supports the hypothesis that quinestrol compromises male fertility by altering sperm membrane composition. A significant decrease in sperm membrane proteins, particularly LDH-C (~35 kDa), was observed following quinestrol treatment. LDH-C is crucial for glycolysis, providing ATP necessary for sperm motility (Farhana & Lappin, 2022) [10] . Its downregulation aligns with previous studies linking impaired LDH-C function to male infertility (Odet et al., 2011; Wang et al., 2013) [24,33] . Furthermore, the significant reduction in the 36 kDa protein band, identified as 17β-hydroxysteroid dehydrogenase (17β-HSD), suggests that quinestrol disrupts androgen biosynthesis, leading to reduced testosterone levels and impaired spermatogenesis (Mindnich et al., 2004; Sidhu et al., 2020) [22, 27] .

The reduction in aromatase (~57 kDa) expression in quinestrol-treated rats is another critical finding. Aromatase plays a vital role in converting androgens to estrogens and is necessary for testicular homeostasis (Carreau et al., 2003).

Previous studies have demonstrated that aromatase inhibition results in impaired sperm function and reduced fertility (Luconi et al., 2002; Rochira & Carani, 2009). The decreased aromatase levels in quinestrol-treated rats further support the hypothesis that quinestrol disrupts the androgenestrogen balance, leading to infertility. Western blot and ELISA analyses confirmed a significant decrease in LDH-C levels in the testis, caput, corpus, and cauda of treated rats. Given LDH-C's exclusive expression in the testis and its essential role in sperm metabolism, its downregulation may serve as a potential mechanism underlying quinestrol-induced infertility. This suggests that LDH-C could be a key target for contraceptive interventions.

Overall, the findings of this study provide strong evidence that quinestrol induces significant disruptions in male reproductive protein expression, affecting critical enzymes, receptors, and sperm maturation proteins. These alterations likely contribute to impaired spermatogenesis, reduced sperm motility, and compromised fertilization ability. Future studies should further explore the molecular mechanisms underlying these disruptions and evaluate the long-term reproductive consequences of quinestrol exposure.

Conclusion

This study demonstrates that quinestrol significantly disrupts key protein expression in the male reproductive system of albino rats.

References

1. Batista M, Rando G, Santini SJ, et al. The role of acrosomal proteins in sperm function and fertilization. Reproductive Biology and Endocrinology. 2022;20(1):45-58.
2. Cavaco JE, Laurentino SS, Gavaia PJ, et al. Estrogen receptors and testicular function: A role for ERα and ERβ in male fertility. Molecular and Cellular Endocrinology. 2009;304(1-2):10-17.
3. Chakraborty S, Saha S. Quinestrol-induced reproductive alterations in wild rodent populations. Journal of Wildlife Research. 2022;48(2):135-147.
4. Cho C, Ge H, Branciforte D, et al. Fertilin β is required for sperm-egg membrane fusion. Nature. 1998;395(6701):402-405.
5. Cilia E, et al. Protein extraction methods in reproductive tissue analysis. Journal of Biochemistry. 2009;256(2):147-153.
6. Colquitt L, Martin J. Hormonal contraceptives: Mechanisms and efficacy. International Journal of Reproductive Medicine. 2017;26(3):215-230.
7. Crowther JR. ELISA: Theory and Practice. Methods in Molecular Biology. 1995;42(1):149-167.
8. Dacheux JL, Dacheux F. New insights into epididymal function in relation to sperm maturation. Reproduction. 2014;147(2):R27-R42.
9. Dhont M. History of oral contraception. European Journal of Contraception & Reproductive Health Care. 2010;15(2):57-66.
10. Farhana A, Lappin SL. Lactate dehydrogenase (LDHC) and its role in sperm metabolism. Journal of Andrology. 2022;43(3):199-211.
11. Gervasi MG, Visconti PE. Molecular changes and signaling pathways involved in sperm capacitation. Journal of Andrology. 2016;37(5):485-498.
12. Gonzalez E, et al. Epididymal secretory proteins and sperm maturation. Journal of Reproductive Fertility. 1984;71(3):501-509.
13. Gundogan M, et al. The role of sperm surface proteins in fertilization. Reproductive Sciences. 2022;29(4):223231.
14. Hess RA, Bunick D, Lee KH, et al. A role for estrogens in the male reproductive system. Nature. 1997;390(6659):509-512.
15. Hubacher D, Trussell J. A definition of modern contraceptive methods. Contraception. 2015;92(5):420421.
16. Hayati A, et al. Oxidative stress and protein oxidation in reproductive tissues. International Journal of Andrology. 2010;33(4):599-610.
17. James ER, et al. Mechanisms of estrogen action in male reproductive health. Endocrinology & Metabolism. 2020;42(4):299-312.
18. Jones R. Plasma membrane modifications during mammalian sperm maturation. International Journal of Andrology. 1998;21(2):45-55.
19. Kiley J, Hammond C. Combined hormonal contraception: Benefits and risks. Obstetrics and Gynecology. 2007;110(5):1373-1380.
20. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685.
21. Leridon H. Demographic perspectives on world population growth. Population Studies. 2020;74(1):116.
22. Mindnich R, Möller G, Adamski J. 17β-Hydroxysteroid dehydrogenases and their role in androgen metabolism. Molecular and Cellular Endocrinology. 2004;218(12):7-20.
23. Naz RK, Vanek N. Role of SP-10 in sperm-egg interaction. Molecular Reproduction and Development. 1998;50(3):316-324.
24. Odet F, Duan C, Willis WD, et al. Expression of the sperm-specific LDH-C in testis and its role in sperm function. Journal of Biological Chemistry. 2011;286(45):40266-40275.
25. Reisse K, et al. Sertoli cell function and its role in spermatogenesis. Biology of Reproduction. 2001;65(3):567-576.
26. Shen S, Wang J, Wang Z, et al. Oxidative stress and its effect on sperm function. Biochemical Journal. 2011;437(1):35-47.
27. Sidhu A, Singh R, Sharma R. Effect of endocrine disruptors on testicular steroidogenesis. Endocrine Research. 2020;45(3):129-141.
28. Sitruk-Ware R, Nath A, Mishell DR. Contraception technology: Past, present, and future. Contraception. 2013;87(3):319-330.
29. Sullivan R, Mieusset R. The human epididymis: Its function in sperm maturation. Human Reproduction Update. 2016;22(3):314-329.
30. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences. 1979;76(9):4350-4354.
31. Turner TT. The epididymis: Sperm maturation, storage, and fertility regulation. International Journal of Andrology. 2008;31(2):195-206.
32. Veron M, et al. Epididymal protein secretion and sperm function. Cell Biology International. 1982;6(2):89-95.
33. Wang Y, Zhang Z, Hu M, et al. Lactate dehydrogenase C and male infertility: A comprehensive review. Human Reproduction Update. 2013;19(5):521-532.
34. Wright KP, et al. New developments in estrogenprogestin contraceptive combinations. Current Medical Research & Opinion. 2020;36(4):651-660.
35. Jones WJ, Leigh JA, Mayer F, Woese CR, Wolfe RS. Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent. Archives of Microbiology. 1983 Dec;136:254-261.