Anat Cell Biol.  2024 Mar;57(1):119-128. 10.5115/acb.23.164.

Glucocorticoids improve sperm performance in physiological and pathological conditions: their role in sperm fight/flight response

• Rago V
• Vivacqua A
• Aquila S

• KMID: 2554246
• DOI: http://doi.org/10.5115/acb.23.164

Abstract

Glucocorticoids play a physiologic role in the adult male reproductive functions, modulating gonadal steroid synthesis and spermatogenesis, through the glucocorticoid receptor (GR). The expression of GR has been described in several key testicular cell types, including somatic cells and early germ cell populations. Nothing is known on GR in human spermatozoa. Herein, we explored the GR expression and its possible role in normal and testicular varicocele semen samples from volunteer donors. After semen parameter evaluation by macro- and microscopic analysis, samples were centrifuged; then spermatozoa and culture media were recovered for further investigations. By western blotting and immunofluorescence analyses we evidenced for the first time in spermatozoa the presence of GR-D3 isoform which was reduced in sperm from varicocele patients. By treating sperm with the synthetic glucocorticoid dexamethasone (DEXA), we found that survival, motility, capacitation, and acrosome reaction were increased in both healthy and varicocele samples. GR involvement in mediating DEXA effects, was confirmed by using the GR inhibitor mifepristone (M2F). Worthy, we also discovered that sperm secretes different cortisol amounts depending on its physio-pathological status, suggesting a defence mechanism to escape the immune system attach in the female genital tract thus maintaining the immune-privilege as in the testis. Collectively, our data suggests a role for glucocorticoids in determining semen quality and function, as well as in participating on sperm immune defensive mechanisms. The novelty of this study may be beneficial and needs to take into account in artificial insemination/drug discovery aimed to enhancing sperm quality.

Keyword

Human sperm; Varicocele; Glucocorticoid receptor; Male infertility

Figure

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  Fig. 1 GR-D3 is present in human ejaculated spermatozoa. Western blot of GR-D3 protein in human sperm from healthy and varicocele patients. MCF-7 extract was used as expression control. N: sperm lysate of tree pooled ejaculates from healthy men; V: sperm lysate of tree pooled ejaculates from patients with varicocele. The experiments were repeated at least four times and the blot shows the results of one representative assay. β-actin was used as loading control. The optical density of the GR-D3 band in N and V samples is reported on the right. GR, glucocorticoid receptor; N, normozoospermic; V, varicocele. ****P<0.001.

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  Fig. 2 Compartimentalization of GR-D3 in human spermatozoa from healthy and varicocele patients. Representative images of immunofluorescence assay in human spermatozoa by using the GR primary antibody (green). Nuclei were counterstained with DAPI (blue). N: sperm of three pooled ejaculates from healthy men; V: sperm of tree pooled ejaculates from patients with varicocele grade III. Scale bars: 12,5 µm. varicocele; N, normozoospermic; V, varicocele.

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  Fig. 3 DEXA induce sperm motility and survival. Sperm motility (A) and survival (B) expressed as percentage of total motile and survival sperm. Columns indicate the mean±SEM of six independent experiments performed in duplicate. N, normozoospermic; V, varicocele; DEXA, dexamethasone. **P<0.05 and ***P<0.01 vs. untreated samples (-).

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  Fig. 4 Dexamethasone (DEXA) increases cholesterol efflux and induces acrosome reaction in sperm. (A) Cholesterol content in culture medium of purified spermatozoa from normal and varicocele samples was measured in the absence (–) or in the presence of the treatments, as indicated. Data are expressed as mg/1×107 sperm. Columns represent means±SEM of six independent experiments carried out in duplicate. *P<0.05, **P<0.02, and ***P<0.0001 vs. untreated sample. (B) Acrosome reaction was analyzed in sperm as indicated. Capacitated sperm (Cap) were used as positive control. Data are expressed as percentage of acrosome reacted sperm. *P<0.01, **P<0.005, and ***P<0.0001 vs. untreated samples (–).

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  Fig. 5 Cortisol secretion is induced by dexamethasone (DEXA) in sperm. Cortisol amount in culture medium of purified spermatozoa from normal and varicocele samples was measured in the presence or not of the treatments, as indicated. Results are expressed as pg/1×107 sperm. *P<0.05 and **P<0.01 vs. untreated sperm (–).

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  Fig. 6 Effect of DEXA on PI3k/AKT and SAPK/JNK pathways. Western blotting analyses were performed in normozoospermic (A) and varicocele sperm (B). Blots show the results of one representative experiment repeated at least four times. β-actin was used as loading control. The optical density is reported on the right. DEXA, dexamethasone; PI3K, PI3-kinase; JNK, c-jun N-terminal kinase; M2F, mifepristone. **P<0.005 vs. untreated sperm (–).

Reference

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