Maturation of sperm volume regulation in the rat epididymis

Abstract

Sperm maturation in the epididymis may involve differences between mature and immature spermatozoa in their volume regulatory osmolyte response. Spermatozoa obtained from the rat caput and cauda epididymidis were examined for their ability to regulate volume after transfer from in situ epididymal osmolality (measured to be 343 +/- 13 and 365 +/- 19 mmol kg(-1), respectively) to that of the female tract in single- and multiple-step protocols. Cells withstood the single-step treatment better than the multistep protocol. Sperm volume estimates by flow cytometric measurements of forward scatter of cells with intact head membranes was more sensitive than those by assessing cell coiling microscopically. At osmolalites below 210 mmol kg(-1) both caput and cauda cells ruptured, limiting the use of flow cytometry. Above this critical value, the use of quinine showed that both caput and cauda cells could regulate volume, but cauda cells were the more effective. Of several organic osmolytes studied, myo-inositol, glutamate and KCl caused only temporary and slight swelling of spermatozoa cells in hypotonic medium. Spermatozoa of both maturities seemed to use potassium as the preferred osmolyte for regulating volume.

Figure 1

Percentages of coiled rat caput (▵▴) and cauda (○•) sperm tails (ordinate: mean ± SEM) during single-step (A) and multistep (B) immersion into decreasing osmolality (abscissa) with (▴•) or without (▵○) 0.8 mmol L⁻¹ quinine (determined by microscopy; n = 4). ^**Significant differences between untreated caput and cauda spermatozoa at osmolalities of 240 mmol kg⁻¹ and below (one-way ANOVA, P ≤ 0.001 [A]; P = 0.006 [B]).

Figure 2

Dot plots (forward scatter ordinate, side scatter abscissa) of spermatozoa from the rat cauda epididymidis during flow cytometry (same sample used with both old [A, C] and new [B, D] protocols) when subjected to single-step hypotonicity (290 mmol kg⁻¹) in the absence (A, B) and presence (C, D) of 0.8 mmol L⁻¹ quinine. Percentages of 5 000 propidium iodide (PI)-negative spermatozoa within each window are given.

Figure 3

Dot plots (forward scatter ordinate, side scatter abscissa) of spermatozoa from the rat caput epididymidis during flow cytometry (same sample used with both old [A, C] and new [B, D] protocols) when subjected to single-step hypotonicity (290 mmol kg⁻¹) in the absence (A, B) and presence (C, D) of 0.8 mmol L⁻¹ quinine. Percentages of 5 000 PI-negative spermatozoa within each window are given.

Figure 4

Dot plots (forward scatter ordinate, side scatter abscissa) of spermatozoa from the rat cauda epididymidis in the absence (A–C) and presence (D–F) of 0.8 mmol L⁻¹ quinine in solutions of single-step decreasing osmolality: 270 mmol kg⁻¹ (A, D), 210 mmol kg⁻¹ (B, E) and 150 mmol kg⁻¹ (C, F). Percentages of 5 000 PI-negative spermatozoa within each window are given.

Figure 5

Dot plots (forward scatter ordinate, side scatter abscissa) of spermatozoa from the rat caput epididymidis in the absence (A–C) and presence (D–F) of 0.8 mmol L⁻¹ quinine in solutions of single-step decreasing osmolality: 270 mmol kg⁻¹ (A, D), 210 mmol kg⁻¹ (B, E) and 150 mmol kg⁻¹ (C, F). Percentages of 5 000 PI-negative spermatozoa within each window are given.

Figure 6

Percentages (ordinate) of swollen (A, B) and PI-positive (C, D) caput (▵▴) and cauda (○•) rat spermatozoa during single-step (A, C) and multistep (B, D) transfer to decreasing osmolality (abscissa) with (▴•) or without (▵○) 0.8 mmol L⁻¹ quinine (from flow cytometric observations; n = 4). ^*Significant differences between quinine-treated and control groups with osmolality for spermatozoa from each epididymal region (one-way ANOVA, caput [A] P = 0.01; caput [B] P = 0.007; cauda [A] P ≤ 0.001; cauda [B] P ≤ 0.001). ^**Significant differences between control values of caput (▵) and cauda (○) spermatozoa with osmolality (one-way RM ANOVA, P = 0.015 [A]; P ≤ 0.001 [B]).

Figure 7

Dot plots (forward scatter ordinate, side scatter abscissa) of spermatozoa from the rat cauda (A, C) and caput (B, D) epididymidis subjected to single-step hypotonicity (290 mmol kg⁻¹) in the presence (A, B) and absence (C, D) of 2 mmol L⁻¹ dithiothreitol (DTT). Percentages of 5 000 PI-negative spermatozoa within each window are given.

Figure 8

Dot plots (forward scatter ordinate, side scatter abscissa) of untreated control (A), 50 mmol L⁻¹myo-inositol-treated (B) and 0.8 mmol L⁻¹ quinine-treated positive control (C) caput epididymidal spermatozoa 15 min after being subjected to single-step hypotonicity (290 mmol kg⁻¹). Percentages of 5 000 PI-negative spermatozoa within each window are given.