Aberrant anogenital distance in XXSxv ('sex-reversed') pseudomale mice

Anogenital distance (AGD). a sex differentiation marker in mice ( Mus musculus ), is thought to be a secondary sexual trait exclusively regulated by androgens during development. However, recent studies suggest that some so-called secondary sexual traits may be influenced by sex-chromosomal genotype. To explore this question further we studied the AGD of the XXSxr 'sex-reversed'mouse, which has adequate androgens for masculinization.
The AGDs of adult and prepubertal XY, XYSxr carrier, XXSxr 'sex-reversed'and XX mice were measured. We found that adult XXSxr 'sex-reversed'mice (which we refer to as pseudomales) and XYSxr carriers have shorter mean AGDs than their XY siblings. Prepubertal XXSxr animals also have shorter mean AGDs than their XY siblings when AGD is expressed as a proportion of body length. XX adult and prepubertal mice have significantly shorter AGDs than the other genotypes at similar stages of development. The differences in AGD between adult and prepubertal XY and XXSxr sibs, and adult XY and XYSxr sibs, reported here, do not appear to be due to androgen levels, since adult testicular and serum testosterone levels are higher in pseudomales and carriers than in XY mice, and fetal pseudomale androgen levels also appear to be at least as high as those of normal males. Furthermore, the AGD differences between genotypes are not correlated to differences in testis size. We conclude that the differences are likely to be due to a tissue specific effect of the genetic constitution of the individuals, as is the case with scrotal development in the marsupial Macropus eugenii (the tammar wallaby).     

Decreased arterial vasculature of the epididymal head in XXSxr pseudomale ('sex-reversed') mice

The microvasculature of epididymides of mature normal XY mice was found to be similar to that reported for other species. The abundant arterial supply to the initial segment is consistent with reports of extensive blood supply associated with high levels of metabolic activity in this segment. We have previously reported the absence of the initial segment in XXSxr pseudomale ('XX sex-reversed') mice. We now present evidence of a decreased arterial supply to the proximal region of the epididymal head in XXSxr pseudomales, including the absence of the dense peritubular microvascular network which surrounds the initial segment in the normal epididymis.     

Epididymides of sex-reversed XX mice lack the initial segment

The genetic factor Sxr causes sex reversal of chromosomally female (XX or XO) mice to phenotypic maleness by inducing development of testes that produce androgens. It has been considered that these sex-reversed animals, called pseudomales, confirm the principle originally developed by Jost that adequate androgenization produces normal phenotypic maleness in mammals, irrespective of chromosomal sex. However, we previously discovered that the epididymis of sex-reversed XX mice (pseudomales of genotype XXSxr) lacks EH 9 cells (epididymal head, cell type No. 9, the "principal cell' of the initial segment). The purpose of the present study was to determine whether cell type EH 9 of XXSxr pseudomales is replaced by a principal cell of a different appearance, or whether the initial segment itself is actually absent. We made serial sections of entire epididymal heads and did microdissections to unravel the highly coiled epididymal duct. Using these two approaches, we studied the sequence of epididymal segments, and estimated lengths of the relevant portion of the epididymal duct; we found that the initial segment of XXSxr pseudomales is truly absent. This is the first report of a mutant genotype causing absence of a segment of the epididymis. The XXSxr mutant appears to be an exception to Jost's principle. This finding shows that, even in full androgenization, male phenotype may not always be independent of chromosomal sex.     

Modulation of the onset of postnatal development of H(+)-ATPase-rich cells by steroid hormones in rat epididymis

Vacuolar type H(+)-ATPase is involved in lumenal acidification of the epididymis. This protein is highly expressed in narrow and clear cells where it is located in the apical pole, and it contributes to proton secretion into the lumen. We have previously shown that in rats, epididymal cells rich in H(+)ATPase appear during postnatal development and reach maximal numbers at 3-4 wk of age. The factors that regulate the appearance of these cells have not been investigated, but androgens, estrogens, or both may be involved. This study examined whether neonatal administration of estrogens (diethylstilbestrol [DES] or ethinyl estradiol) or an antiandrogen (flutamide), or the suppression of androgen production via administration of a GnRH antagonist (GnRHa), was able to alter the appearance of cells rich in H(+)-ATPase in the rat epididymis when assessed at age 25 days. Surprisingly, all of these treatments were able to significantly reduce the number of H(+)-ATPase positive cells; this was determined by immunofluorescence and confirmed by Western blotting. In contrast, neonatal coadministration of DES and testosterone maintained the expression of H(+)-ATPase in the epididymis at Day 25 despite the high level of concomitant estrogen exposure. These findings indicate that androgens, acting via the androgen receptor, are essential for the normal development of epididymal cells rich in H(+)-ATPase, and that treatments that interfere directly or indirectly with androgen production (GnRHa, DES) or action (flutamide, DES) will result in reduced expression of H(+)-ATPase. Our findings do not exclude the possibility that estrogens can directly suppress the postnatal development of cells in the epididymis that are rich in H(+)-ATPase, but if this is the case, this suppression can be prevented by testosterone administration.     

Developmental changes in and hormonal regulation of estrogen and androgen receptors present in the rabbit epididymis

Both androgen and estrogen receptors (AR and ER) are present in the rabbit epididymis. We have used the sucrose gradient method to examine receptor sedimentation properties, receptor concentration, and distribution of receptors among the caput, corpus, and cauda of the epididymis to determine changes that occur in these parameters as the animals age. The 9S form of the ER is present in all three epididymal segments of the immature rabbit, with the highest concentration occurring in the cauda. The 8.2S form of the AR is also present in all three segments of the immature epididymis, with the highest concentration occurring in the caput. Short-term castration (3 days) leads to an increase in the amount of both AR and ER detected. ER are present in all segments of the immature epididymis at higher concentrations than AR. The functional 9S form of the ER disappears as the animals mature, the result of a tissue-specific protease that our laboratory previously has shown proteolyzes ER to a non-DNA-binding 3.8S form. Long-term castration (3 mo) of adult rabbits results in the reappearance of the 9S form of the ER in all segments of the epididymis. The reappearance of the 9S form of the ER is also seen in animals castrated for 1 mo, but not in those castrated for 2 wk. Administration of testosterone once daily for 2 wk to adult animals castrated for 6 wk results in the disappearance of the 9S form of the ER and the reappearance of the 3.8S form, suggesting that the tissue-specific protease is androgen-dependent. In this way, circulating androgens may play a role in regulating the concentration and form of the ER in the rabbit epididymis. There is little change in the concentration or distribution of AR in the epididymis of adult rabbits castrated for 3 mo as compared to those castrated for 3 days. This implies that circulating androgens are not required for maintenance of AR in the epididymis. Our data demonstrate that there are temporal differences in the presence and concentration of ER and AR in the epididymis and suggest that there is a differential, age-dependent regulation of the development and function of the epididymis by androgens and estrogens.     

Ornithine decarboxylase activity as a marker of androgen and antiandrogen action in the rat epididymis

After castration, there was a marked decrease in serum androgen concentration at 6 h, and a dramatic inhibition of ornithine decarboxylase (ODC) at 12 h. Administration of testosterone propionate to castrated rats at a dose of 0.05 mg/animal restored ODC activity to the normal value. However, no change was observed when intact rats were treated with testosterone even at a 40-fold higher dose, indicating that endogenous androgens present in intact rats are far in excess for maintenance of maximal levels of activity. Administration of the antiandrogen flutamide to intact rats caused a moderate decrease in epididymal weight, whereas this effect was more pronounced in castrated, androgen-treated rats. In the latter, the effect of flutamide was significant at the lowest dose used (0.5 mg/day). ODC activity was significantly decreased by flutamide treatment of intact rats, but even at the highest dose used (10 mg/day) only a 39% inhibition was observed. In flutamide-treated rats, LH concentrations were markedly increased, as were serum and epididymal androgens. In androgen-treated castrated rats, flutamide caused epididymal ODC to fall to undetectable values. These results show that: (1) androgens are essential for the maintenance of ODC activity in the epididymis; (2) epididymal ODC activity is maximally stimulated by endogenous androgens, at least in the pubertal rat; (3) the apparent potency of flutamide is substantially lowered by an increase in epididymal androgens. We suggest that ODC is a sensitive marker of the action of androgens and antiandrogens in the epididymis.     

Epididymal phenotype in luteinizing hormone receptor knockout animals and its response to testosterone replacement therapy

Previous studies reported that epididymis contains functional LH receptors. The LH receptor knockout mice, which have epididymal phenotypes, gave us an opportunity to test the hypothesis that testosterone replacement alone may not be sufficient to reverse phenotypes to wild-type epididymis. The morphological phenotype in knockout animals includes a decrease in luminal diameter of the proximal and distal caput and cauda epididymis, the absence of clear and halo cells in the epithelial lining, a decrease in the height of principal cells and the number of cells containing cilia, a decrease in cilia length, and a change from basal to central location of nuclei in the principal cells. The biochemical phenotype includes a decrease in periodic acid-Schiff reaction product, reflecting the glycogen and glycoprotein synthesis and secretion, a decrease in androgen receptor (AR) and estrogen receptor (ER)beta, and an increase in ERalpha levels. Twenty-one-day testosterone replacement therapy in 30-day-old knockout animals reversed some, but not all, morphological and biochemical phenotypes. Those that did not reverse include luminal diameters of proximal and distal caput and cauda epididymis, the percentage of ciliated principal cells in caput epididymis, and nuclear AR localization. In summary, while our results reaffirm that androgens are important for normal epididymal morphology and function, they indicate that LH could be required for certain facets of epididymal morphology and/or function.     

Physiology of the epididymis and spermatozoa

The epididymis is an ideal extragonadal target site to inhibit fertility in the male. Synthesis and secretion of constituents like sialic acids, protein and glycerylphosphoryl choline by the epididymal epithelium under androgen control provide an ideal fluid environment for sperm maturation. An optimal level of sialic acid secretion by the epididymal epithelium is needed to maintain functional integrity of sperm. The existence of specific androgen receptors in the epididymis and spermatozoa are related to their ability to metabolise androgens.     

Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice

Low molecular weight, acid-stable proteinase inhibitors from epididymal and seminal vesicle homogenates were isolated and characterized. The isolation procedure consisted of gel filtration, trypsin affinity, and ion exchange chromatography. The inhibitor from seminal vesicle homogenates has a molecular weight of approximately 6,200, and that of the epididymal inhibitor was estimated at 4,000. Antiserum directed against the seminal vesicle inhibitor did not react with epididymal components. The epididymal inhibitor shows competitive, whereas the seminal vesicle inhibitor shows noncompetitive inhibition against trypsin on double reciprocal plots. Both inhibitors are effective against trypsin and acrosin but not against chymotrypsin, kallikrein, thrombin, or plasmin. To verify site of origin and to investigate androgen dependency of the epididymal inhibitor, mice were efferentiectomized, orchiectomized, or orchiectomized with androgen supplementation. Gel filtration profiles of acid-treated epididymal homogenates from normal and efferentiectomized animals show inhibitor peaks in the same regions. The concentration of acid-stable inhibitor from epididymal homogenates decreased with orchiectomy but returned to normal values when exogenous androgen was supplied. These observations suggest that the low molecular weight inhibitor in the epididymal homogenates is distinct from that in the seminal vesicles. Furthermore, the inhibitor associated with epididymal homogenates is androgen-dependent, and the epididymis is the site of origin of this inhibitor.     

Androgenic regulation of enzymes involved in DNA synthesis in epididymis of young rats.

In the epididymis of young rats, activities of DNA polymerases alpha, beta and gamma and DNA topoisomerase I decreased after castration. DNA polymerase alpha and gamma increased with androgen administration and activity reached 81.3% and 78.0%, respectively, of the activity in the sham-operated group on day 21. Activity of DNA polymerase beta remained at the activity of day 7 during androgen administration and was almost the same as that in the sham-operated group on day 21. DNA topoisomerase I activity showed a slight increase with androgen administration and reached 50.3% of that in the sham-operated group. The activities of these enzymes were not fully restored to those in the sham-operated group. These results indicate that in young rats activities of epididymal DNA polymerase alpha and gamma and DNA topoisomerase I are partially, and that of DNA polymerase beta wholly, dependent on androgens and may provide a means of investigating the regulation of epididymal cell proliferation.     

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis

Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80-90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50-60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification.     

Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice

We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.     

Evidence for high levels of androgen in peripheral plasma during postnatal development in a marsupial: the gray short-tailed opossum (Monodelphis domestica).

Plasma samples were assayed for androgen in gray short-tailed opossums (Monodelphis domestica) on the day of birth and at selected ages through adulthood. Levels of androgen in mixed-sex plasma pools of animals 4 and 8 days of age were higher than in either sex at all other ages examined. At postnatal Days 16, 30, and 60 (weaning), levels of androgen were equivalent in males and females and as high as in adult males. In both sexes, androgen levels were lower at postnatal Day 84 (juveniles) than at younger ages; after puberty, levels were significantly higher in males than in females. These findings are discussed with respect to similarities and differences between marsupials and eutherians in hormonal environment during the perinatal period and with respect to the possible role of androgens in sexual differentiation of the gray opossum brain.     

Tissue androgens and androphilic proteins in rat epididymis during sexual development

The appearance of the epididymal 8s cytoplasmic receptor in the rat during sexual development was followed and correlated with the endogenous concentrations of three biologically active androgens in the epididymis, testosterone (T), dihydrotestosterone (DHT)and 5α-androstane-3α, 17β-diol (Diol).The results indicate that receptors could not be evidenced in the 10-day-old animal in which androgens were undetectable. The 8s receptor was first detected in the 20 day-old rat, coincidently with a rise in the concentration of DHT (0,24 ng/ epididymis) while T and Diol remained too low to be measured. At the 26th day of life a peak of androgen binding activity, sedimenting at approximately 4S, was seen and probably corresponds to ABP. This pattern of two binding peaks (8 and 4S) remained constant through adulthood. The association constant (Ka) of the 8s receptor from 35-, 45- and 60-day-old rats was found to be similar.T became detectable in the epididymides of the 35 day-old rat (0.03 ng/ep.) and increased with sexual maturation. However, DHT concentrations were higher than those of T at all ages studied and Diol could not be detected at any age.These results raise the possibility that the synthesis or availability of receptors in the developing animals is under androgenic control.     

Regulation of epididymal function and sperm maturation--endocrine approach to fertility control in male.

The structural and functional integrity of the epididymis, the acquisition of fertilizing ability by spermatozoa and their viability within the epididymis are androgen dependent phenomena. Although the precise mechanism by which sperm maturation and viability in the epididymis are brought about by androgen are not clearly understood, it is generally held that specific epididymal secretions produced under the influence of androgen affect these events. Though the spermatozoa appear to remain viable in a low androgen environment, sperm maturation requires a relatively high androgen environment. Against this background the potentiality of antiandrogens as extragonadal antifertility agents has been discussed. Studies with steroidal and nonsteroidal antiandrogens have revealed that in adult animals the secretory activity of the epididymis, as evidenced by the level of glycerylphosphorylcholine, either remains unaffected or is stimulated under their influence. These studies have further indicated that the extragonadal antifertility action of antiandrogens will depend upon their ability to (1) lower the testicular androgen synthesis and/or androgen binding protein, which possibly serves as a carrier of androgen from the testis to epididymis; (2) to lower local androgen synthesis as a result of reduced levels of circulating androgen, and (3) to inhibit 5 alpha-reduction of testosterone to dihydrotestosterone and/or to inhibit androgen binding to receptors. Success in the rational development of new antifertility agents for male which will act by controlling epididymal function will depend upon a clear understanding of the factors that regulate epididymal secretion and the role of epididymal secretions in sperm maturation and survival.     

Sex determining genes and vitellogenin synthesis in Drosophila melanogaster

Summary This study investigates the relationship between sexual phenotype and ability to synthesize vitellogenin (yolk proteins, YPs) in Drosophila. Various mutations were used to transform XX and XY animals into intersexes or pseudomales (Table 1). The presence or absence of YPs in the haemolymph and in the fat body was determined by SDS gel electrophoresis, fluorography, and precipitation of YPs with anit-YP antibody (see Fig. 1). YPs were synthesized whenever the flies displayed at least some female morphological characteristics, regardless of their sex chromosome constitution (Table 1; Fig. 2). Pseudomales (definition see p. 1) did not produce detectable amounts of YPs despite their female XX-karyotype. Immature ovaries, transplanted into adult males or pseudomales, developed normally and synthesized YPs, but the fat bodies of the host males or pseudomales were not induced to synthesize YPs. Vitellogenesis was, however, induced in the fat bodies of males and pseudomales by injection of 20-hydroxyecdysone (ecdysterone) (Fig. 3). The results are interpreted to mean that the sexual pathways are controlled by a small number of key genes that regulate the synthetic activities of many sex-specific genes. However, the female-specific YP genes can be activated with ecdysterone although the genetic signals are set for male differentiation.     

Differentiation of the rat epididymis after withdrawal of androgen

Summary The differentiation capacity of the rat epididymis after depletion of androgen was studied in organ culture and in castrated rats. The differentiation of narrow cells in 5- and 10-day-old explants and in 10-day-old castrated rats suggests that: (i) the testicular androgens are not essential for their differentiation, (ii) a differential androgen dependence exists among the epididymal cell types, (iii) the undifferentiated epithelial cells are the precursors of the narrow cells.