Testicular migration, spermatogenesis, temperature regulation and environment of the sheath-tail bat, Taphozous georgianus

The testes of the common sheath-tail bat of tropical Australia undergo a seasonal migration between the abdomen and the scrotal pouches, while each cauda epididymidis is permanently maintained in the scrotal pouch. Straps of smooth muscle attach to both the cranial and caudal poles of the testes, and these extend cranially to the diaphragm and caudally to the cauda epididymidis. The testicular arteries are not coiled. Among the environmental factors investigated, maximum temperature correlated most significantly with testicular descent, and the number of spermatogonia per bat also correlated most significantly with maximum temperature. Body temperature of a captive bat ranged from 25 to 38 degrees C and this was closely related to body weight and ambient temperature. It seems likely that the scrotal pouch provides a temperature slightly below that of the body and so facilitates sperm storage in the permanently scrotal cauda epididymidis. Migration of the testes probably serves to ameliorate the seasonal temperature fluctuations to which they are exposed while the relatively high correlation between maximum environment temperature and spermatogonial numbers suggests that temperature may be a proximate influence on reproduction in the sheath-tail bat.     

The influence of body temperature and castration on the protein composition of fluid in the rat cauda epididymidis

Polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and immunoelectrophoretic analysis, revealed that several characteristic proteins disappear from the luminal fluid of the rat cauda epididymidis when it is maintained at body temperature. On SDS-PAGE gels prepared under reducing conditions, one Coomassie-blue staining band of Mr 18,000 disappeared and another of 52,000 was significantly reduced after only 6 days; bands of Mr 23,000, several in the Mr 34-38,000 range, one of Mr 48,000, and others of Mr 100-200,000 were eliminated or markedly reduced after 15 days at body temperature. Some were glycoprotein, as judged by their affinity for FITC-ConA. At 15 days after castration there was a broadly similar but rather more extensive disappearance of macromolecules, and of glycoproteins in particular, from caudal fluid. The fact that several similar proteins are diminished in or disappear from fluid or the cauda epididymidis maintained at body temperature, or after androgen withdrawal, raises the possibility that one or more such proteins play a role in sperm storage there.     

Regionalized expression of CD52 in rat epididymis is related to mRNA poly(A) tail length

The regional pattern of CD52 expression in the rat epididymis was followed by Northern analyses and carbohydrate-labelling of glycoconjugates on Western blots. CD52 mRNA showed a novel aspect of regionalization, namely region-dependent length differences in its poly(A) tail. ‘Short’ CD52 mRNA molecules were present in all parts of this organ and also in the seminal vesicles. Additionally, the cauda epididymidis contained mRNA molecules with an extended poly(A) tail. Their appearance coincided with the occurance of the principal M_r ≈ 26 kDa glycopeptide in the cauda region, representing the CD52 product. CD52 expression seemed to be regulated or modulated synergistically by androgens, temperature, and (an) unknown testicular factor(s), depending on the poly(A) tail length of its mRNA. Androgens alone exerted an effect only on molecules with ‘short’ poly(A) tails. They were down-regulated in castrated animals, and restored to normal levels upon testosterone supplementation. However, ‘long’ CD52 mRNA molecules were not affected. Only if combined with the exposure of the epididymis to the elevated temperature of the abdomen, castration of animals resulted in a complete loss of the CD52 mRNA, including the ‘long’ cauda species. Loss of ‘long’ CD52 mRNA molecules was also observed when the abdominal location was combined with efferent duct ligation. This combination of treatments, however, did not affect ‘short’ CD52 mRNA levels. Loss of the ‘long’ CD52 mRNA molecules by any treatment coincided with a loss of the principal M_r ≈ 26 kDa glycopeptide from caudal protein extracts. Mol. Reprod. Dev. 48:433–441, 1997. © 1997 Wiley-Liss, Inc.     

Epididymal specificity and androgen regulation of rat EP2

In primates, expression of the EP2 gene is androgen-dependent and epididymis-specific. EP2 mRNA expression was investigated in caput, corpus, and cauda regions of rat epididymis and in 15 other rat tissues. Polymerase chain reaction and Northern analyses showed that rat EP2 is expressed predominantly in the proximal caput epididymidis. EP2 mRNA expression was determined in proximal epididymides from castrated, sham-operated, and efferent duct-ligated rats. In castrated rats, EP2 mRNA decreased to <10% of that in sham-operated rats between Days 3 and 4 postcastration, demonstrating the androgen dependence of EP2 expression. In epididymides ligated unilaterally at the efferent ducts, EP2 mRNA levels were approximately equal to those in the unligated contralateral epididymides or in sham-operated rats, indicating that EP2 expression does not depend on testicular factors. In bilaterally castrated rats, immediate and delayed testosterone replacement showed the dependence of EP2 expression on circulating androgens. Injection of testosterone propionate (TP) on Days 0, 1, 2, and 3 postcastration maintained EP2 mRNA levels approximately equal to those in sham-operated rats. Starting at Day 4 postcastration, daily injection of TP for 7 days restored EP2 mRNA to approximately normal levels. These data indicate for the rat that EP2 is expressed specifically in the proximal caput epididymidis and that its expression depends on circulating androgens but not on testicular factors.     

Androgenic regulation of messenger RNA in rat epididymis

1. The regulation by testosterone of mRNA complexity and mRNA activity was investigated in rat caput and cauda epididymidis. 2. The sequence complexity of cytoplasmic poly(A)-containing RNA from normal rats was determined by homologous hybridization with radiolabelled complementary DNA probes by using RNA in excess. Computer analysis of results suggested that hybridization could best be described by curves composed of two components distinguished by their relative abundance. Thus caput-epididymidal RNA consists of approx. 260 moderately abundant and 16400 scarce sequences, whereas cauda-epididymidal RNA consists of approx. 124 moderately abundant and 13400 scarce sequences. Judging by heterologous-hybridization reactions, castration did not result in appreciable alterations in either sequence complexity or the relative abundance of the two classes of poly(A)-containing RNA. 3. To investigate if individual mRNA sequences were regulated by androgens, mRNA was translated in a cell-free system derived from reticulocyte lysate. Since most of the translation products had a different mobility on sodium dodecyl sulphate/polyacrylamide gels from the authentic proteins synthesized in tissue minces, antibodies were used to identify specific translation products. Antibodies to the two related major proteins (mol.wt. 18500 and 19000) secreted by the caput epididymidis and whose synthesis is stimulated by testosterone both precipitated a single translation product of mol.wt. 21000. That this polypeptide was a precursor to the secreted proteins was suggested by the fact that the addition of microsomal membranes isolated from dog pancreas resulted in the appearance of a polypeptide of mol. wt. 19000. 4. Translation of RNA from the caput epididymidis of rats of different hormonal status showed that mRNA activity for the 21000-dalton polypeptide declined after castration, but could be restored by treating rats with testosterone. 5. It is concluded that testosterone stimulates the synthesis of a major protein secreted by the caput epididymidis by regulating its mRNA activity.     

Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (³⁵S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein-specific in that only selected proteins became associated with the cells. Binding was not cell-specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.     

Characterization and hormonal regulation of protein synthesis by the murine epididymis

Protein synthesis and secretion were examined in vitro by incubating minced tissue with [35S]methionine. The incorporation of label into tissue plus medium was linear for the 5 h of incubation. The percentage of available label incorporated into protein increased with the weight of tissue used. Approximately 13% of the label incorporated appeared in the medium after 5 h of incubation. Release of radioactive protein into the medium was characterized by an initial slow release (1-2 h) followed by a more rapid linear release between 3 and 5 h. Polyacrylamide gel electrophoresis revealed that the pattern of radioactive proteins present in the medium was different from and less complex than the tissue proteins. Substantial differences in protein patterns from different epididymal regions could be detected. The caput epididymidis was particularly active in secreting proteins characteristic of this region, whereas the corpus and cauda synthesized and secreted similar proteins. At least one of these proteins characteristic of the caput is stabilized by disulphide bonds. Short-term (9 day) castration resulted in reduced synthesis and secretion of several of these epididymal proteins. Testosterone administered after 9 days of castration reinitiated synthesis of some but not all of these epididymal proteins.     

Glutathione-independent prostaglandin D2 synthase in ram and stallion epididymal fluids: origin and regulation

Microsequencing after two-dimensional electrophoresis revealed a major protein, glutathione-independent prostaglandin D2 synthase (PGDS) in the anterior epididymal region fluid of the ram and stallion. In this epididymal region, PGDS was a polymorphic compound with a molecular mass around 30 kDa and a range of pI from 4 to 7. PGDS represented 15% and 8% of the total luminal proteins present in this region in the ram and stallion, respectively. The secretion of the protein as judged by in vitro biosynthesis, and the presence of its mRNA as studied by Northern blot analysis, were limited to the proximal caput epididymidis. Using a specific polyclonal antibody raised against a synthetic peptide, PGDS was found throughout the epididymis, decreasing in concentration toward the cauda region. PGDS was also detected in the testicular fluid and seminal plasma by Western blotting. Castration and efferent duct ligation in the ram led to a decrease in PGDS mRNA and secretion. PGDS mRNA was not detected in the stallion 1 mo after castration, and it was restored by testosterone supplementation. This study showed that PGDS is present in the environment of spermatozoa throughout the male genital tract. Its function in the maturation and/or protection of spermatozoa is unknown.     

Transport of carnitine by the epididymis of the cynomolgus macaque (Macaca fascicularis)

Unravelled tubules from the monkey caput and cauda epididymidis were perfused through the lumen in vitro during immersion in an organ bath kept at scrotal temperature and containing [3H]carnitine and [14C]inulin. The specific transport of carnitine from the bath to the lumen was constant for 4 h and reached a steady-state value of about 90 pmol/30 min per cm perfused length in the cauda and about 30 pmol/30 min/cm in the caput. These regional variations in carnitine transport differ from those found in the rat epididymis but may be relevant to human epididymal physiology.     

Effects of castration, 5 alpha-dihydrotestosterone and cyproterone acetate on enzyme activity in the mouse epididymis

The influence of castration, androgen replacement therapy and cyproterone acetate on the activity of beta-glucuronidase, acid and alkaline phosphatases and glucose-6-phosphate dehydrogenase was studied in the caput, corpus and cauda epididymidis of the mouse. The results add further evidence that the epididymis is not uniform but has regional differences in activity. Thus beta-glucuronidase was found to be androgen-dependent only in the cauda epididymidis, whereas glucose-6-phosphate dehydrogenase was under androgenic control in the caput epididymidis. The response of alkaline and acid phosphatases to castration and to androgen replacement was different in different segments.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.     

Lower temperature of the cauda epididymidis facilitates the storage of sperm by enhancing oxygen availability

The temperature coefficients for the oxygen consumption rate of sperm from the rat, mouse, bull, and sea urchin were measured to be within a range of 2.7 to 3.0. For a reduction in temperature from 37°C to 30°C, the respiration rate of mammalian sperm declined by a factor of approximately 2.1. Hence, the availability of oxygen to sustain these sperm substantially increased over this temperature range. In addition, the solubility of oxygen in cauda epididymidal fluid increased by 7.6% for a decline in temperature from 37°C to 30°C. The increased availability of oxygen to sperm at the lower temperature is related to the increased storage capacity of the cauda epididymidis in scrotal mammals. In this context, it is suggested that the evolutionary migration of the cauda epididymidis to a cooler, extra-abdominal location may have been influenced by the increased availability of oxygen to sperm and hence resulted in an increased capacity to sustain and thereby store more sperm per unit volume of duct.     

Alterations of gene expression in potato (Solanum commersonii) during cold acclimation

Plantlets of Solanum commersonii stem-culture were acclimated at 5°C day/night temperature for 14 days. Cold hardiness increased from – 3.5°C to – 8.6°C. During the course of acclimation, the synthesis of polypeptides was investigated and poly (A⁺) RNA was isolated. Translation products of poly(A⁺) RNA in a rabbit rcticulocyte lysate system were then analyzed. During the 14 days of acclimation, 23 cold-induced polypeptides were identified. Most of them disappeared following 1 day of de-acclimation at a 20/15°C day/night regime. The synthesis of one group of polypeptides is prominent and stable throughout the acclimation period. The other group is transient. The most prominent and stable polypeptides have molecular weights of 21, 22, 31 and 83 kDa.
Acclimation alters translatable mRNA population during the development of cold hardiness. Two mRNAs encoding in vitro translation products at 26 and 27 kDa were identified during the course of acclimation. These proteins may play important roles in the overall programming for the development of cold hardiness in tuber-bearing S. commersonii.     

Ultrastructure of the perfused rat epididymis: Effect of luminal sodium ion concentration

Summary The appearance of the rat epididymal epithelium changed when it was perfused in vivo through the lumen with unphysiologically high sodium ion concentrations; dilatation of intercellular spaces (ICS) at threshold concentrations of 30mM-Na⁺ in the cauda and about 55mM-Na⁺ in the corpus was associated with absorption of water from the lumen. Despite the distended ICS, junctional complexes appeared intact, and their integrity was confirmed by the exclusion of luminal horseradish peroxidase (HRP) from the ICS, and by demonstrating that circulating [³H]inulin did not enter the lumen. Smooth ER and lipid droplets in the principal cells of the corpus epididymidis were well maintained, and the preservation of granular ER in principal cells of the cauda epididymidis lent morphological support to the continued secretion of protein in this segment. However, occasional distension or involution of inner Golgi cisternae was evident in principal cells after 3–6 h perfusion. In contrast to multivesicular bodies of principal cells, the apical and basal vacuoles characteristic of clear cells changed in size with different perfusing solutions. When low Na⁺ concentrations were perfused large translucent vacuoles were frequently found in the apical cytoplasm of clear cells in the corpus and cauda epididymidis, and filled vacuoles became larger and showed a decrease in content density in the cauda epididymidis. These large vacuoles were absent from tissue perfused with high Na⁺ concentrations. Normal pinocytotic activity of both cell types was demonstrated by perfusing HRP which was taken up by the normal route in principal cells, with some transfer to the Golgi cisternae. By far the most HRP was accumulated in clear cell vacuoles irrespective of the composition of the perfusing solution.     

Interactions of labeled epididymal secretory proteins with spermatozoa after injection of 35S-methionine in the mouse

The sequential interactions of epididymal secretory proteins with spermatozoa during epididymal transit were examined. Mice received injections of 35S-methionine, and the radiolabeled luminal fluid and sperm-associated proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis at various times after injection. The majority of the luminal fluid and sperm-associated proteins were found in the caput epididymidis at 8 h; by 7 days, many of these proteins had been transported to the cauda epididymidis. Two classes of epididymal protein-sperm interactions were distinguished on the basis of regional synthesis and secretion. The major class consisted of proteins that were synthesized, secreted, and bound to spermatozoa in the caput epididymidis. In this class, however, the binding of proteins to the spermatozoa was variable. For example, a protein of 25 kDa remained associated with spermatozoa in substantial amounts during epididymal transit, while proteins of 40 and 35 kDa decreased in amount. Other proteins such as a protein of 18 kDa did not remain associated with spermatozoa. Another class of proteins (54, 44, 29 kDa) were synthesized and secreted from all epididymal regions but bound only to caput spermatozoa. Most of the epididymal proteins appeared to be tightly bound to the spermatozoa since spermatozoa already saturated with the unlabeled protein in the distal epididymis remained so even though the spermatozoa were surrounded by labeled proteins in the luminal fluid. These studies demonstrate that a variety of specific interactions occur between epididymal secretory proteins and spermatozoa as they migrate and mature in the epididymis.     

Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis.

(35S) methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells have been analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with (35S)methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of six early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent molecular weights of 75,000 (75K), 42K, 21K, 18K, 15K, and 11K. Supportive data were obtained by coelectrophoresis of (35S)- and (3H)methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides have not been previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately twofold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization.     

Influence of abdominal temperature and luminal contents on the cauda epididymidis of the rat

In an attempt to determine if changes previously described in the epididymides of cryptorchid testes were related to the elevated environmental temperature or to the absence of normal luminal constituents, rats were divided into four test groups. Group I animals were made unilaterally cryptorchid. Animals in Group II had only the cauda epididymidis of one side maintained within the abdominal cavity (cryptepididymal) while the caput epididymides and testes remained in the scrotum. The testes of animals in Group III remained in the scrotum but had their efferent tubules ligated on one side. Testes of unoperated rats and contralateral testes of the test animals served as controls. The histochemical demonstration of sorbitol dehydrogenase (SDH) was used to determine differences in functional activity and light and electron microscopy were used to determine structural changes. SDH activity could not be demonstrated in the cauda epididymidis of cryptorchid and efferent tubule-ligated animals; animals in which the luminal contents were obviously changed. These same groups of animals showed abnormal folding of the basal surface of the epididymal epithelium at the ultrastructural level. Activity of SDH could be demonstrated in control epididymides and in those that contained normal luminal contents but were maintained at the temperature of the abdominal cavity. The basal surface of the epididymal epithelium was not unusual in these animals. The results indicate that the epididymis is influenced to a greater extent by changes in luminal contents than by temperature elevation.     

A function for pericardial cells in an insect

The pericardial cells (PCs) of fifth instar Calpodes ethlius larvae are functionally adapted for filtering hemolymph and sequestering and digesting proteins. They also have a structure appropriate for the synthesis of proteins for secretion. PC secretion has been investigated by labelling the cells with [³⁵S]methionine ti vitro with detection of newly synthesized polypeptides appearing in the medium by electrophoresis and fluorography. Sources possibly contributing to the appearance of newly synthesized polypeptides in the medium, such as cell breakdown and fat body contamination have been ruled out. The post-incubation medium of PCs contains at least six newly synthesized polypeptides. Three of these polypeptides, having relative molecular masses of 82, 57 and 43 kDa, react with antibodies to hemolymph. At least one additional polypeptide is similar by two-dimensional analysis to that naturally present in hemolymph. PCs incubated together with the heart to which they are normally attached, secrete additional polypeptides that are presumed to come from the heart. The 82 kDa polypeptide secreted by the PCs is similar to the subunits of arylphorin secreted by fat body and other tissues. We conclude that PCs secrete proteins into the hemolymph although the amount may be small relative to that of the fat body.     

Embryonic protein composition and synthesis during imbibition of white lupin seeds: effect of low temperatures

White lupin seed imbibition under low temperature conditions (8 °C) and their subsequent effects on embryonic protein composition and synthesis were investigated. The response to low temperatures was accompanied by changes in polypeptide composition and synthesis. The embryonic axes labelled in vivo with (³⁵S)-methionine retained their capacity to synthesize proteins during imbibition at 8 °C. The synthesis of some polypeptides was increased during low temperature treatment as compared to that at 25 °C. These cold-induced polypeptides were essentially detected in a molecular mass range from 15 to 35 kDa and a pI range from 6.3 to 8.7.     

Two fibrinogen-like proteins,FGL1 and FGL2 are disulfide-linked subunits of oligomers that specifically bind nonviable spermatozoa

Nevertheless, a nonviable sperm population is present in the cauda epididymidis of many species. Degenerating spermatozoa release enzymes that could have detrimental effects on the viability of neighboring cells, and they are source of autoantigens that induce an autoimmune response if they escape the blood-epididymis barrier. Does the epididymis have specialized protective mechanism(s) to segregate the viable sperm population from defective spermatozoa? Previously, we identified a fibrinogen-like protein-2 (fgl2) that specifically binds to and polymerizes into a cocoon-like complex coating defective spermatozoa and sperm fragments. The objective of the present study is to identify the subunit composition of the fgl2-containing oligomers both in the soluble and cocoon-like complex. Our proteomic studies indicate that the 260/280 kDa oligomers (termed eFGL) contain two distinct disulfide-linked subunits; 64 kDa fgl2 and 33 kDa fgl1. Utilizing a PCR-based cloning strategy, the 33 kDa polypeptide has been identified as fibrinogen-like protein-1 (fgl1). Immunocytochemical studies revealed that fgl1 selectively binds to defective spermatozoa in the cauda epididymidis. Northern blot analysis and in situ hybridization demonstrated the high expression of fgl1 in the principal cells of the proximal cauda epididymidis. Co-immunoprecipitation analyses of cauda epididymal fluid, using anti-fgl2, demonstrate that both fgl1 and fgl2 are present in the soluble eFGL. Our study is the first to show an association of fgl1 and fgl2 both in the soluble and in the sperm-associated eFGL. We conclude that our results provide new insights into the mechanisms by which the potentially unique epididymal protein functions in the recognition and elimination of defective spermatozoa.