The cytoplasmic droplet of rat epididymal spermatozoa contains saccular elements with Golgi characteristics.

The cytoplasmic droplet of epididymal spermatozoa is a small localized outpouching of cytoplasm of the tail of unknown significance. EM revealed flattened saccular elements as the near exclusive membranous component of the droplet. Light and electron microscopic immunolabeling for Golgi/TGN markers showed these saccules to be reactive for antibodies to TGN38, protein affinity-purified alpha 2,6 sialyltransferase, and anti-human beta 1,4 galactosyltransferase. The saccules were isolated by subcellular fractionation and antibodies raised against this fraction immunolabeled the saccules of the droplet in situ as well as the Golgi region of somatic epithelial cells lining the epididymis. The isolated droplet fraction was enriched in galactosyltransferase and sialyltransferase activities, and endogenous glycosylation assays identified the modification of several endogenous glycopeptides. EM lectin staining in situ demonstrated galactose and N-acetyl galactosamine constituents in the saccules. Endocytic studies with cationic and anionic ferritin as well as HRP failed to identify the saccules as components of the endocytic apparatus. Epididymal spermatozoa were devoid of markers for the ER as well as the Golgi-associated coatamer protein beta-COP. It is therefore unlikely that the saccular elements of the droplet participate in vesicular protein transport. However, the identification of Golgi/TGN glycosylating activities in the saccules may be related to plasma membrane modifications which occur during epididymal sperm maturation.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum androgens and steroidogenic enzyme activities in the male rat reproductive tract.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to impair reproductive function of males in animal models, possibly due to a reduction in serum androgen levels. Thus, TCDD may alter the testosterone biosynthetic pathway in the testis or the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) in androgen target tissues. Pregnant Sprague Dawley rats were gavaged with TCDD (0, 0.2 or 1.0 microg/kg) on day 15 of gestation only. TCDD caused a reduction in the body weight gain of the dams in both dose groups and a significant reduction in litter size in the higher dose group. Litters delivered normally and TCDD exposed male offspring grew at the same rate as controls. Males were sacrificed at 15, 30, 45, 60, 90 and 120 d of age. Steroidogenic enzyme activities were determined in testicular microsomes and androgen target tissue nuclear fractions. Serum androgens were measured by radioimmunoassay (RIA). At 30 d of age, rats exposed to 1.0 microg/kg TCDD exhibited lower 17-hydroxylase activity (P < 0.05) and lower caput-corpus epididymal weights (P < 0.05). At 45 d of age, the same treatment resulted in testicular 3beta-HSD, 17beta-HSD and 5alpha-reductase activities that were significantly greater (P < 0.05) but, conversely, serum androgens were one quarter the values evident in controls (P < 0.05). At the other ages, no differences were observed in serum androgens and, with the exception of lower 17beta-HSD activity at 90 d of age (P < 0.05), no other differences in testicular steroidogenic enzyme activities were found. 5Alpha-reductase activities in the androgen target tissues were also unchanged. Histological examination of testes showed that the spermatogenic profile was identical to controls at all ages.

Biogenesis of specialized cytoskeletal elements of rat spermatozoa.

Our results on the formation of the ODF and perforatorium are diagrammatically summarized in Figures 30 and 31. The developmental expression of proteins making up these two cytoskeletal elements differs in timing, duration and intracellular localization. The ODF proteins are synthesized exclusively during the latter part of spermiogenesis, well after transcriptional activity in the haploid germ cell nucleus has ended. This implies that these major integral proteins of the tail are translationally regulated and that mechanisms must exist for the storage and eventual release of the mRNAs encoding these proteins. The perforatorial proteins, on the other hand, begin to be synthesized during the meiotic prophase reaching a peak of production in early spermiogenesis just before the initiation of the condensation of the spermatid's nucleus, at which time RNA synthesis stops. Another major difference between ODF and perforatorial protein production is that there seems to be a coordinated activity between the synthesis and the assembly of the ODF proteins, whereas there appears to be an almost 25 day delay between the initial meiotic synthesis and final condensation of perforatorial proteins in the subacrosomal space at the end of spermiogenesis. As for the intracellular localization of ODF and perforatorial proteins both have unprecedented distributions. The ODF proteins appear to be concentrated in a particular type of granular body which is especially abundant in the elongated spermatid at the time of peak ODF assembly. The perforatorial proteins, on the other hand, appear to be concentrated in the nuclei of pachytene spermatocytes and round spermatids until their displacement into the cytoplasm during nuclear condensation. Both forms of localization suggest a storage role for these proteins uniquely adapted by the spermatid to regulate the assemblies of the respective cytoskeletal elements.

Distribution and possible role of perinuclear theca proteins during bovine spermiogenesis.

The perinuclear theca (PT) is a unique cytoskeletal element that encapsulates the mammalian sperm nucleus. It is divided into subacrosomal and postacrosomal regions. The objective of this study was to analyze and stage the intracellular distribution of several prominent bull PT proteins during spermatogenesis. For this purpose, polyclonal antibodies raised and affinity-purified against the 15.5-, 25-, 28-, 32-, 36-, and 60-kDa bull PT polypeptides were used as probes on sections of aldehyde-fixed testes. Immunoperoxidase staining revealed that the PT polypeptides first appeared early in spermiogenesis, concomitant with early steps of development of the acrosomic system. Immunogold labeling further showed that these polypeptides were peripherally associated with the entire acrosomal membrane, before and during the attachment of the acrosomic vesicle onto the spermatid's nucleus. Once the acrosome had capped the nucleus the labeling resided mainly in the subacrosomal region of the spermatid, between the inner acrosomal membrane and nuclear envelope. Later, during the elongation of the spermatid's nucleus, the labeling with all antibodies except the anti-15.5-kDa antibody extended caudally over the assembling postacrosomal sheath. This study suggests that the perinuclear theca proteins play an instrumental role in the attachment, spreading, and binding of the acrosome onto the nucleus of spermatids.

Androgen binding protein secretion and endocytosis by principal cells in the adult rat epididymis and during postnatal development.

Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.

Molecular cloning and developmental expression of the major fibrous sheath protein (FS 75) of rat sperm.

The fibrous sheath (FS) is a cytoskeletal structure that encases the axoneme in the principal piece of the spermatozoon tail. In the rat, it is composed of several proteins, of which a 75-kDa polypeptide (FS 75), as estimated by PAGE, is the most prominent. The objectives of this study were to clone and sequence this protein and to characterize its transcriptional and translational origins during spermatogenesis. Initially, we isolated two overlapping cDNA segments that encoded a large part of the FS 75 protein but lacked the initiation codon for translation. Both clones were obtained by screening a rat testicular phagemid cDNA library with an anti-FS 75 polyclonal antibody. An upstream portion of the FS 75 mRNA containing the initiation codon was obtained by polymerase chain reaction with a pair of specific primers. Accounting for the overlap in all segments, 2786 nt of an approximately 3-kb FS 75 mRNA was obtained. The amino acid sequence of the longest possible open reading frame of the rat FS 75 was found to be similar to two previously cloned variants of the major FS polypeptide of mouse spermatozoa. Sequence analysis of the rat FS cDNA revealed anchoring kinase A protein domains and several kinase phosphorylation sites, supporting the idea that this protein plays a crucial role in the motility of spermatozoa. The presence of a potential N-myristoylation site suggests that this protein may covalently bind to the inner leaflet of the plasma membrane (PM), which in turn may explain the close association of the FS and PM from early development. Developmental northern blot analysis and in situ hybridization revealed that the FS 75 mRNA is haploid expressed, with an abundant level of mRNA in late round spermatids. Maximum levels of the FS 75 polypeptide, as determined by immunocytochemistry, correlated with a rapid decline in corresponding mRNA levels in step 14-16 spermatids. Since transcription termination occurs several steps earlier, the bulk of FS 75 mRNA appears to be translationally regulated.

Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa.

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.

Occurrence and formation of cytoskeletal proteins in mammalian spermatozoa.

Mammalian spermatozoa are composed of specialized cytoskeletal elements, which appear to have no structural or protein counterparts in somatic cells. Most evident are the outer dense fibres (ODF) and fibrous sheath (FS) of the sperm tail and the perinuclear theca (PT) of the sperm head. The purpose of this study is to review our results on the occurrence and assembly of proteins making up these three elements during spermatogenesis. Our approach was to raise antibodies against the prominent proteins of these elements and to immunolocalize them on testicular sections prepared for histological and ultrastructural analyses. We found that all of the cytoskeletal proteins considered were expressed exclusively during the haploid phase of development and that the proteins of each element had similar if not identical patterns of expression. The PT proteins were synthesized in the first half of spermiogenesis and were associated with acrosome formation, while the ODF and FS proteins were synthesized in the second half of spermiogenesis. The ODF proteins assembled in a proximal-distal direction along the length of the axoneme, while the FS proteins assembled in the opposite direction; both assemblies eventually meeting and overlapping within the periaxonemal cytoplasmic compartment. During assembly the ODF proteins appeared to be temporarily stored in granulated bodies of the cytoplasmic lobe, while the FS proteins were randomly distributed throughout the cytoplasm. In the case of the PT, there appeared to be an interdependence between PT assembly and acrosome formation. The developmental protein distribution patterns observed for each of the elements suggest unique cellular targeting mechanisms adapted by the spermatid to regulate the assemblies of the respective cytoskeletal proteins.

Protein composition of the perinuclear theca of bull spermatozoa.

The perinuclear theca is a cytoskeletal structure that covers the nucleus of mammalian spermatozoa except for a narrow zone around the insertion of the tail. It shows two distinct regions, a subacrosomal layer or perforatorium and, continuing caudally beyond the acrosomic system, the postacrosomal sheath. The main objective of this study was to analyze the protein composition of the bull perinuclear theca and the distribution of some of its proteins. The perinuclear theca was extracted from demembranated and acrosome-depleted sperm heads by alkaline treatment. Gel electrophoresis revealed that the extract was composed of over 20 polypeptide bands, of which the 15.5-, 25-, 28-, 32-, 36-, and 60-kDa bands were most prominent. Polyclonal antibodies prepared against these major polypeptides were screened on Western blots of the theca extracts and were immunolocalized on sections of spermatozoa prepared for electron microscopy. Antibodies against the 15.5- and 60-kDa polypeptides reacted monospecifically with their respective bands on Western blots, and immunogold labeled the perforatorium and the entire perinuclear theca, respectively. Antibodies against the 25-, 28-, 32-, and 36-kDa polypeptides showed distinctive patterns of cross-reactivity with other polypeptides on Western blots, but immunogold labeled exclusively the entire perinuclear theca. In conclusion, this study provides evidence that the protein composition of the bull perinuclear theca is complex: at least six major polypeptides make up this cytoskeletal element. As to their distribution, the 15.5-kDa polypeptide is localized exclusively to the subacrosomal layer, and the other five major polypeptides analyzed in this study appear to be shared between the subacrosomal and post-acrosomal regions of the perinuclear theca.

Origin and distribution of perforatorial proteins during spermatogenesis of the rat: an immunocytochemical study.

The perforatorium is the subacrosomal portion of the perinuclear theca that encapsulates the nucleus of spermatozoa. In the rat, the perforatorium is a curved pointed structure, which in cross section is triangular in outline over the apical half and beyond the tip of the nucleus. The perforatorium, composed of several proteins, appears as a distinct structural entity only at the very end of spermiogenesis. In this study, polyclonal antibodies prepared against the entire isolated perforatorial fraction and against the major 16 and 34 kDa perforatorial polypeptides were used to determine the distribution of perforatorial proteins in germinal cells at various steps of differentiation. Immunoperoxidase staining at the LM level and quantitative immunogold labeling at the EM level were used. The labeling patterns with all three antibody preparations were identical. The immunolabeling first appeared in early pachytene spermatocytes and increased progressively, with a statistically significant upward trend, in both the nuclei and cytoplasm of spermatocytes and spermatids until step 9 of spermiogenesis. Up to this step the labeling concentration was significantly higher over the nucleus than over the cytoplasm. During nuclear condensation in steps 9 and 12 spermatids, there was a progressive loss of all the labeling over the nucleus and a corresponding increase of labeling over the cytoplasm. During steps 16-18, the early signs of condensation of perforatorial proteins occurred next to the inner acrosomal membrane. Then during step 19 there was a sudden condensation of perforatorial proteins into a definitive perforatorium. Thus proteins destined to form this cytoskeletal structure reside in both the nucleus and cytoplasm of spermatocytes and spermatids until nuclear condensation of the latter. Thereafter, they are restricted to the spermatid's cytoplasm and finally condense around the elongated nucleus at the end of spermiogenesis.

Isolation, structure and protein composition of the perforatorium of rat spermatozoa.

Heads of spermatozoa were sonically separated from tails and treated in 1 N NaOH until the perforatoria were partially detached from the nucleus. Their complete detachment was then assured by repeatedly passing the suspension through a 22-gauge needle. The perforatoria were then separated from nuclei on sucrose gradients and the purity of the fraction was verified by electron microscopy. The isolated perforatoria were denatured and used to raise antibodies or run on polycrylamide gels. Such gels revealed many polypeptide bands, six of which were most prominent (Mr approximately 13,000, 13,400, 16,000, 33,000, 35,000, and 43,000). Of these, the 16,000 Mr polypeptide was major. Anti-perforatorium serum reacted with the perforatoria of fixed spermatozoa, with a substance found between the plasmalemma and the outer acrosomal membrane of the acrosomal head cap and with the inner component of the ventral spur, but not with the postacrosomal dense lamina. This observation indicated that the perforatorium and dense lamina, although structurally continuous to form the perinuclear theca, are biochemically distinct. On Western blots, the anti-perforatorium serum reacted with the prominent polypeptides of the perforatorium and cross-reacted with some less prominent polypeptides of the fibrous sheath (FS) and outer dense fibers (ODF), most notably with a 16,000 Mr polypeptide found in both. Likewise anti-FS or anti-ODF serum cross-reacted with some major and minor polypeptides of the perforatorium, again most notably with a major 16,000 Mr polypeptide. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. These results demonstrate that the perforatorium is composed of several polypeptides, is immunologically distinct from the postacrosomal dense lamina, may be immunologically similar to a substance found between the plasmalemma and outer acrosomal membrane and to a substance found on the inner aspect of the ventral spur, has antigenic determinants in common with the FS and ODF, and may share a 16,000 Mr polypeptide with these two cytoskeletal structures of the flagellum.

Light microscopic immunocytochemical study of fibrous sheath and outer dense fiber formation in the rat spermatid.

The formation of the fibrous sheath (FS) and outer dense fibers (ODF), two major cytoskeletal components of the tail of spermatozoa, was analyzed in the seminiferous epithelium by immunoperoxidase techniques applied to paraffin-embedded testicular sections. Antibodies were prepared from purified FS and ODF fractions and from major 75 and 14.4 kDa FS polypeptides and major 32-26 14.4 kDa ODF polypeptides. The immunostaining results showed that the production of FS and ODF proteins appeared to be exclusive to step 9-19 spermatids and lasted over the duration of a full cycle of the seminiferous epithelium, or 12.8 days. During this period there was seemingly an initial lag of short duration between the synthesis and assembly of FS and ODF proteins followed by a long process of coordinated activity. Peak cytoplasmic immunoreactivity was reached in step 15 for FS proteins and midstep 16 for ODF proteins and remained elevated thereafter for approximately 80 hr for both FS and ODF proteins. The immunoreactivity was more uniform and diffused for FS proteins and granulated or clumpy for ODF proteins. Assembly of FS proteins along the axoneme proceeded in a distal to proximal direction while for ODF proteins assembly proceeded in a proximal to distal direction. The main route of elimination of residual cytoplasmic FS and ODF proteins appeared to take place through the cytoplasmic droplets and residual bodies, respectively. There appeared to be no variation in step reactivity between the major ODF polypeptides tested and only minor variation in step reactivity between the major FS polypeptides tested. However, although the 14.4 kDa polypeptides of FS and ODF share antigenic determinants, they do not appear to be identical, because they presented different immunolocalizations during spermiogenesis and different directions of assembly along the axoneme.

Comparison of the effects of gossypol, estradiol-17 beta and testosterone compensation on male rat reproductive organs.

The effects of gossypol acetic acid (GAA) were compared with those induced by estradiol-17 beta (E2), testosterone, and a combination of these steroids. GAA was administered s.c. to adult rats at doses of 25 to 30 mg . kg BW . day for 30 days, while steroids in polydimethylsiloxane tubing of various lengths were implanted s.c. for 30 days or longer. GAA and E2 at the doses used had similar effects: they caused a graded atrophy of sex organs, discriminative degeneration of spermatogenic cells, impairment of Sertoli cells, decrease in serum testosterone, reduction in androgen receptor binding and retardation in body growth. Supplementing GAA and E2 treatments with 14-cm testosterone implants had a counteracting effect on organ weight losses: seminal vesicles recovered above, ventral prostate within and epididymides below control values, whereas the testes did not respond. The organs most refractory to supplementation therapy were those in which GAA and E2 were most effective in depressing androgen receptor binding. Aside from having similar antiandrogenic effects as E2 and other steroids, GAA induced a specific flagellar syndrome which testosterone therapy could not prevent, indicating that this action is hormonally independent.

Segmental aplasia of the mitochondrial sheath and sequelae induced by gossypol in rat spermatozoa.

The effect of gossypol upon organelles of rat sperm was investigated by light and electron microscopy. The drug was administered s.c. for 2 to 30 days at 20 mg/kg BW/day. Sperm from the testis, caput, corpus, and cauda epididymis were examined at regular intervals, during and after treatment, for periods extending up to 70 days. The drug induced a specific effect in the sperm tail. It consisted of segmental aplasia of the mitochondrial sheath observed in high incidence in testicular and epididymal sperm. This primary lesion, in the authors' view, predisposed a development of secondary lesions as sperm advanced along the epididymis. Secondary lesions included bulging, dislocating, fraying, or breaking of axial fibers, bending or breaking of the tail, and decapitation. A minimum of 3 days of treatment was necessary to produce an effect above control values, while 9 days or longer induced lesions in almost all sperm. Motility ceased with 30 days of treatment. Fertilizing capacity was inversely related to the increase and decline of lesions.

The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat.

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.

Developmental expression of immobilin in the rat epididymis.

BACKGROUND: Immobilin is a protein secreted by principal cells of the distal initial segment, intermediate zone and caput epididymidis of adult rats, which serves to immobilize spermatozoa. In the distal cauda, epithelial clear cells are involved in its endocytosis. The objective of this study was to correlate the developmental events in the maturation of the epididymis with the timing of immobilin secretion and endocytosis in order to evaluate the testicular or epididymal factors which may influence or regulate immobilin expression. METHODS: Our approach was to follow and compare the developmental expression of immobilin by light microscope immunocytochemistry in control and efferent duct ligated rats of different postnatal ages. RESULTS: Coincident with the morphological maturation of the principal cells by postnatal day 39, immobilin displayed the characteristic secretory immunostaining pattern found in adults. This adult-like expression occurred despite the absence of spermatozoa in the lumen but was coincident with high levels of circulating and luminal androgens. In contrast, immobilin secretion in rats whose efferent ducts were ligated at day 15 was weak to non-existent in the principal cells of the caput epididymidis at day 28 and remained so into adulthood, indicating that principal cells of this region of the epididymis are dependent either directly or indirectly upon testicular factors present in the lumen for immobilin expression. However, secretion of immobilin in the principal cells of the distal initial segment was unaffected by ligation and unlike the case in control rats high levels of immobilin also continued to be secreted into adulthood by the principal cells of the proximal initial segment. Thus in the distal initial segment immobilin secretion is not regulated by luminal factors originating from the testis, while in the proximal initial segment the normal suppression of immobilin that occurs by postnatal day 39 is. Despite ligation, endocytosis of immobilin by clear cells of the distal cauda epididymidis occurred by day 49, indicating that luminal testicular factors are not essential for stimulating the uptake of immobilin by these cells. CONCLUSIONS: The results taken together suggest that there are stimulatory and inhibitory luminal testicular factors involved in the regional development of immobilin secretion in the epididymis. There are also immobilin secreting regions in the epididymis, whose secretory development is independent of luminal testicular factors.

Developmental expression of sulfated glycoprotein-2 in the epididymis of the rat.

BACKGROUND: Sulfated glycoprotein-2 (SGP-2), also designated as clusterin, is a protein secreted by the epididymis and which binds to spermatozoa. In adult rats it is secreted at high levels by principal cells of the distal initial segment, intermediate zone and caput epididymidis, and at relatively lower levels by principal cells of the corpus and cauda epididymidis. The objective of this study was to correlate the developmental events in the maturation of the epididymis with the timing of SGP-2 expression in order to evaluate the testicular or epididymal factors which may regulate it. METHODS: Our approach was to follow and compare the developmental expression of SGP-2 by immunocytochemistry in normal untreated control rats and rats whose efferent ducts were ligated on day 15 and examined at different postnatal ages thereafter. RESULTS: In control animals, SGP-2 expression in principal cells of the distal initial segment, intermediate zone, and caput and distal cauda epididymidis, as characterized in normal 90-day-old adult animals, was attained between postnatal days 39 and 49. However, only by postnatal day 56 did SGP-2 display in the corpus and proximal cauda the characteristic secretory pattern found in adult rats. In contrast, in efferent duct ligated rats examined at postnatal day 64, SGP-2 was absent in principal cells of the corpus and proximal cauda epididymidis but continued to be secreted by the distal initial segment, intermediate zone, and caput and distal cauda epididymidis. Furthermore, unlike the case in control rats, SGP-2 was secreted at high levels by the principal cells of the proximal initial segment. Thus during normal postnatal development, in the proximal initial segment, the production of SGP-2 is suppressed by luminal factors originating from the testis, while in the distal initial segment, intermediate zone, and caput epididymidis, it is unaffected by these factors. On the other hand, the production of SGP-2 in the corpus and proximal region of the cauda epididymidis is normally stimulated by luminal factors originating from the testis, while in the distal cauda, it is unaffected by these factors. CONCLUSIONS: Our results thus show a differential regulation of SGP-2 expression in principal cells of the proximal versus distal regions of the epididymis and even within subdivisions of each region. In some regions of the epididymis, SGP-2 production appears to be unaffected by luminal factors originating from the testis, while in other regions it is either inhibited or stimulated by these factors.