Efferent Duct Fluid Research Articles

Absence of Estrogen Receptor Alpha Leads to Physiological Alterations in the Mouse Epididymis and Consequent Defects in Sperm Function1

Male mice deficient in ESR1 (ERalpha) (Esr1KO mice) are infertile, and sperm recovered from the cauda epididymis exhibit reduced motility and fail to fertilize eggs in vitro. These effects on sperm appear to result from defective epididymal function and not a direct effect on spermatogenesis, as Esr1KO germ cells transplanted into wild-type testes yield normal offspring. We hypothesized that the previously described defect in efferent duct fluid reabsorption would lead to alterations in the epididymal fluid milieu, which would negatively impact sperm function. Analysis of the epididymal fluid revealed that the Esr1KO maintains a higher luminal pH throughout the epididymis, confirming an inability of the efferent ducts and/or epididymis to properly acidify the luminal contents. Subsequent studies showed that these abnormalities were not the result of global defects in epididymal function since protein secretion by the Esr1KO epididymis appeared normal as judged by SDS-PAGE of total secreted proteins and by immunoblotting of candidate secreted proteins. To gain insight into the basis of the aberrant fluid homeostasis in the Esr1KO epididymis, the expression of several enzymes and transporters known to be involved in acid/base regulation were analyzed. The levels of SLC9A3 (NHE3) as well as carbonic anhydrase XIV and SLC4A4 (NBC1) were all reduced in the proximal portion of the Esr1KO epididymis, while other components appeared unaffected, including other ion transporters and ATP6V0A1 (V-ATPase). The altered luminal milieu of the Esr1KO epididymis was shown to lead to a corresponding increase in the intracellular pH of Esr1KO sperm, relative to sperm from control animals. Since pH and bicarbonate ions are critical regulators of sperm cAMP levels and motility, we attempted to bypass the abnormal luminal and intracellular environment by supplementing sperm with exogenous cAMP. This treatment rescued all defective motility parameters, as assayed by CASA, further showing that motility defects are not intrinsic to the sperm but, rather, result from the abnormal epididymal milieu.

Adrenal independence of fluid and electrolyte reabsorption in the ductuli efferentes testis of the rat.

The ductuli efferentes testis (efferent ducts) of the rat were studied to determine whether fluid and electrolyte reabsorption by the ducts is under the control of adrenal mineralocorticoids. Testicular fluid output and the rate of fluid reabsorption by the ducts were determined in sham-operated controls and in rats which had been adrenalectomized 10 days previously, adrenalectomized 10 days previously and given aldosterone therapy (75 micrograms kg-1 day-1 S.C.) from day 3 to day 10 post adrenalectomy, and in rats given injections of the aldosterone antagonist, spironolactone, for 10 days (10 mg kg-1 day-1 S.C.). The values for testicular fluid output and fluid flow from the efferent ducts (means +/- S.E.M.) in the sham-operated rats were 36.0 +/- 7.8 and 1.23 +/- 0.12 microliters h-1, respectively, resulting in an estimate of fluid reabsorption by the efferent ducts of 94.8 +/- 1.6% of the testicular fluid output. None of the treatments resulted in a significant change in testicular fluid output or in fluid reabsorption from the efferent ducts. Similarly, the treatments did not significantly alter the osmolality or electrolyte concentrations in fluid samples from the rete testis or the distal end of the efferent ducts (sham-operated values for rete testis and efferent duct fluid, respectively, were: osmolality, 311.2 +/- 1.7 and 302.7 +/- 5.7 mosmol kg-1; [Na+], 151.2 +/- 5.6 and 150.8 +/- 8.1 mmol l-1; [Cl-], 147.9 +/- 6.6 and 126.7 +/- 2.2 mmol l-1; [K+], 15.2 +/- 0.7 and 13.5 +/- 2.4 mmol l-1). It is concluded that, like the homologous proximal tubule of the metanephric kidney, fluid reabsorption from the efferent ducts is independent of adrenal mineralocorticoid control.

Ontogeny and cellular origin of an interleukin-1-like factor in the reproductive tract of the male rat.

We have recently isolated an interleukin-1 (IL-1)-like factor from the rat testis, which originates from the seminiferous tubules and is a protein with an MW of 17,000 and a pI of 5-6. This paper reports on the appearance of the IL-1-like factor during postnatal development and investigates its cellular origin further. IL-1 activity was measured by a murine thymocyte proliferation assay. Very low IL-1 activity was present in culture medium conditioned by seminiferous tubules from rats aged 10 or 20 days. From 30 days of age, increasing amounts were detected, reaching a maximum level in adult animals (60-90 days). No IL-1 activity was found in medium conditioned by peritubular cells. Sertoli cell-enriched seminiferous tubules obtained from experimentally cryptorchid or from prenatally irradiated rats produced much higher levels of IL-1 activity than did those obtained from intact testes. IL-1 activity was detected in efferent duct fluid after ligation of the efferent ducts for 24 h, indicating that the IL-1-like factor was secreted into the tubular lumen. Low levels of IL-1 activity were detected in extracts of epididymal tissue and epididymal sperm, whereas ejaculated seminal plasma, seminal vesicle fluid and extracts of seminal vesicles (together with the coagulating glands) and ventral and dorsolateral prostate lacked IL-1 activity. Instead, seminal plasma inhibited testicular IL-1 activity dose-dependently without affecting cell viability in the thymocyte cultures. Although its biological function remains to be defined, our results indicate that the testicular IL-1-like factor is produced by Sertoli cells and that its appearance during development coincides with the initiation of active spermatogenesis in the rat testis.

Testicular degeneration in a male goat. A case report

Semen samples from an 18-month-old infertile Lamancha buck contained less than 10% motile spermatozoa and a high incidence of morphological abnormalities. Degenerate detached heads, tightly coiled tails, and thickened midpieces were the prominent morphological defects, affecting 46%, 30%, and 15% of ejaculated spermatozoa, respectively. At necropsy, the diagnosis of testicular degeneration was made, characterized by focal, noninflammatory seminiferous tubule occlusion and mineralization, with a more generalized sloughing of tubular germinal epithelium. When examining spermatozoa morphology from various sites of the excurrent ducts, detached heads were readily seen in efferent duct fluid (> 30% incidence) and the coiled tails and other midpiece defects became prominent with passage through the caput epididymis.

Distribution of testosterone and 5alpha-dihydrotestosterone in rat epididymis and their concentrations in efferent duct fluid.

5alpha-Dihydrotestosterone concentrations were found to be appreciably higher in the proximal portion of the rat epididymis than in the distal portion. Following ligation of the efferent ducts of a testis, the 5alpha-dihydrotestosterone concentration diminished in the proximal half but not in the distal half of the epididymis. Epididymal fluid and spermatozoa were washed out from minces of epididymal tissue. The numbers of spermatozoa present in the wash fluid and in the homogenate of washed minces of epididymal tissue were used as measures for the amounts of epididymal fluid present in these fractions. Of the total amount of 5alpha-dihydrotestosterone (30-2 ng/g tissue) in the proximal portion, 20-5 ng were localized in the epididymal fluid, whereas of the total amount of 5alpha-dihydrotestosterone (8-6 ng/g tissue) in the distal portion, 2-8 ng were found in the epididymal fluid. The amounts of testosterone present in the fluids of both proximal and distal portions were 1-2 ng/g tissue. In contrast to the epididymal fluid, efferent duct fluid had a high concentration of testosterone (28-8 ng/ml) and a low concentration of 5alpha-dihydrotestosterone (1-9 ng/ml). These data suggest that the fluid surrounding the spermatozoa in the testis and the epididymis contains a high concentration of androgen and that as the fluid moves from the testis to the epididymis there is a clear change in the ratio of testosterone to 5alpha-dihydrotestosterone.

Immunological cross reactivity between testicular androgen-binding protein and serum testosterone-binding globulin.

WE have reported the presence of a testicular androgen-binding protein (ABP) which is secreted into the testicular fluid of several mammalian species1. ABP is produced by the Sertoli cells and is specifically stimulated by follicle stimulating hormone (FSH)1–7. It is found in particularly high concentrations (3×10−7 M) in the efferent duct fluid of rabbits.

Testicular androgen-binding protein (ABP): comparison of abp in rabbit testis and epididymis with a similar androgen-binding protein (TeBG) in rabbit serum

Testicular androgen-binding proteins (ABP) in rabbit testis, caput epididymis and efferent duct fluid (EDF) were compared to a similar androgen-binding protein TeBG) in rabbit serum. The affinity of these proteins for 5α-dihydrotestosterone (DHT) at 0 °C ( K a ABP = 1.6 × 10 9 M −1 and K a TeBG = 1.9 × 10 9 M −1) and their steroid specificities were similar (DHT > androstanediol > progesterone and androstenedione). ABP and TeBG had also almost identical Stokes radii (42.8 ± 1.2 and 43.9 ± 0.8 Å, respectively), sedimentation coefficients (4.7 ± 0.2 S and 4.4 ± 0.2 S, respectively) and electrophoretic mobility ( R f = 0.4 in 6 1 2 % polyacrylamide gels). Calculation of molecular weights from Stokes radii and sedimentation rates indicated a molecular weight of 74,000 (69,000–78,000) for TeBG and 76,000 (71,000–82,000) for ABP. The corresponding frictional ratios were 1.61 for TeBG and 1.55 for ABP assuming a partial specific volume (– v) of 0.70 cm 3/g. Polyacrylamide gel electrophoresis (PAGE) at different gel concentrations gave a mean molecular radius of 2.74 nm, also indicating a molecular weight of about 75,000 (– v = 0.70 cm 3/g). ABP and TeBG could not be separated by PAGE; however, partial separation of ABP and TeBG was achieved by isoelectric focusing and ion-exchange chromatography on DEAE-cellulose. TeBG focused at pH 5.4, whereas ABP formed a distinct peak of bound radioactivity at pH 4.7. Also by ion-exchange chromatography, ABP in both testis and epididymal supernatants was shown to have an apparently higher surface charge than TeBG in rabbit serum. The concentration of ABP in efferent duct fluid (2 × 10 −7 M = 60 pmol/mg protein) was much higher than TeBG in male rabbit serum (5.2 × 10 −8 M = 0.7 pmol/mg protein). These findings ruled against the possibility that ABP in the testis and epididymis could have been derived directly from serum. It is concluded that ABP and TeBG are very similar if not identical proteins both serving as transport and carrier proteins in their respective compartments.

Sertoli cell origin of testicular androgen-binding protein (ABP)

In this report it is suggested that the specific androgen-binding protein (ABP), previously shown to originate in the testis of rat and other species, is produced by the Sertoli cells. This suggestion is based upon the following experimental findings: 1) ABP was found in high concentrations in testicular efferent duct fluid but only in trace amounts in inter-tubular lymph. 2) ABP could be recovered from crude preparations of testis tubules, but not from Leydig cells from the same testes. 3) Testes whose germinal epithelium had been severely damaged by gamma irradiation showed no decrease in ABP content. The transport of ABP to epididymis was also preserved as judged from the levels of ABP in caput epididymis. 4) Testes that were completely devoid of germ cells following prenatal gamma irradiation showed high levels of ABP. These high levels approached zero following hypophysectomy, but could be restored by FSH administration to the hypophysectomized animals. ABP has been well characterized and now provides a valuable experimental tool as an indicator of Sertoli cell function.

Concentration of steroid hormones and androgen binding protein (ABP) in rabbit efferent duct fluid.

Androgenic hormones are necessary for the completion of normal spermatogenesis (1) and may mediate the stimulatory influence of the gonadotrophins FSH and LH on germ cell development (2). Although the stimulatory effects of LH on Leydig cell secretion are well documented, the mechanism by which FSH influences the various testicular compartments has been more speculative. The demonstration of a high affinity androgen binding protein (ABP) in the testicular efferent duct fluid (EDF) of the rat (3,4) and rabbit (5) and its control by FSH (2,6,7) has raised the possibility that FSH may influence the local concentration of androgens in the immediate vicinity of the developing and maturing germ cells. In order to test this hypothesis, a radioimmunoassay technique, which enables the simultaneous measurement of 8 steroids, including the major androgens, (8,9) was applied to a study of the steroid concentrations in rabbit EDF. It was hoped that steroid assays in conjunction with ABP measurements in the same samples would answer the following questions: (a) What is the steroid composition of the fluid bathing the testicular sperm? (b) Is there a relationship between the concentration of androgens and the levels of ABP?

Demonstraion of an adrogen binding protein (ABP) in rabbit testis: Secretion in efferent duct fluid and passage into epididymis

An androgen binding protein (called ABP) is present in 105,000 g av supernatants of rabbit testis and caput epididymidis but not in cauda. Testicular efferent duct fluid contains ABP in a concentration of 1.5 × 10 −7M, assuming one binding site per molecule of protein. Ligation of the efferent ducts causes ABP to increase in testis supernatant and disappear from supernatant of caput epididymidis. An androgen binding protein similar to ABP is present in rabbit serum; however, the concentrations of ABP per mg protein are much higher in efferent duct fluid and caput supernatant than in serum. These findings indicate that ABP is secreted in the testicular efferent duct fluid and may serve as a carrier of androgen from testis to epididymis.

A high-affinity androgen-binding protein (ABP) in rat testis: evidence for secretion into efferent duct fluid and absorption by epididymis.

An androgen-binding protein (ABP) was detected in 105,000 × g supernatants of rat testis homogenates after removal of endogenous steroids by charcoal extraction. The ABP was identical to that previously demonstrated in highspeed supernatants of epididymis homogenates. When the exocrine secretion of the testis was blocked by ligation of efferent ducts, ABP increased in testis supernatant in proportion to the retention of testicular fluid while at the same time it disappeared from epididymis supernatant, first from the caput and later from the cauda. Treatment with large doses of testosterone did not alter the disappearance from epididymis. ABP disappeared almost completely from caput supernatant within three days after efferent duct ligation. When efferent ducts and corpus epididymidis were ligated simultaneously to block the flow of luminal fluid into and out of the caput, the disappearance of ABP from caput supernatant was only partially prevented. This finding suggested that a major portion of ABP is ei...

Androgen-binding protein in efferent duct fluid of rat testis.

Testicular fluid obtained from the rete testis and vasa efferentia has been reported to contain testosterone in concentrations approaching the amount in spermatic venous blood (Voglmayr, Waites & Setchell, 1966; White & Hudson, 1968). Flow rates, which have been established in several mammalian species (Setchell, 1970; Tuck, Setchell, Waites & Young, 1970) would indicate that the exocrine secretion may deliver a substantial quantity of androgen to the caput epididymidis. Testosterone and possibly other androgens in rete testis fluid might also exert a direct influence on spermatozoa as they are transported in this fluid. In the present report, a specific androgen-binding protein with high affinity for dihydrotestosterone (DHT) and testosterone (T) is demonstrated in rete testis and efferent duct fluid from the rat testis. Fluid