Functional importance of palmitoyl protein thioesterase 1 (PPT1) expression by Sertoli cells in mediating cholesterol metabolism and maintenance of sperm quality

Sertoli cells are a type of nurse cell in the seminiferous epithelium that are crucial for sustaining spermatogenesis by extending nutritional and energy support to the developing germ cells. Dysfunction of Sertoli cells could cause disordered spermatogenesis and reduced fertility in males. In this study, we focused on the expression and function of palmitoyl protein thioesterase 1 (PPT1), a lysosomal depalmitoylating enzyme, in Sertoli cells. Here, we show that PPT1 expression in Sertoli cells is responsive to cholesterol treatment and that specific knockout of Ppt1 in Sertoli cells causes male subfertility associated with poor sperm quality and a high ratio of sperm deformity. Specifically, Ppt1 deficiency leads to poor cell variably accompanied with abnormal lysosome accumulation and increased cholesterol levels in Sertoli cells. Further, Ppt1 deficiency results in poor adhesion of developing germ cells to Sertoli cells in the seminiferous epithelium, which is likely to be responsible for the reduced male fertility as a consequence of declines in sperm count and motility as well as a high incidence of sperm head deformity. In summary, PPT1 affects sperm quality and male fertility through regulating lysosomal function and cholesterol metabolism in Sertoli cells.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Disrupted sperm function and fertilin beta processing in mice deficient in the inositol polyphosphate 5-phosphatase Inpp5b.

Inpp5b is an ubiquitously expressed type II inositol polyphosphate 5-phosphatase. We have disrupted the Inpp5b gene in mice and found that homozygous mutant males are infertile. Here we examine the causes for the infertility in detail. We demonstrate that sperm from Inpp5b(-/-) males have reduced motility and reduced ability to fertilize eggs, although capacitation and acrosome exocytosis appear to be normal. In addition, fertilin beta, a sperm surface protein involved in sperm-egg membrane interactions that is normally proteolytically processed during sperm transit through the epididymis, showed reduced levels of processing in the Inpp5b(-/-) animals. Inpp5b was expressed in the Sertoli cells and epididymis and at low levels in the developing germ cells; however, mice lacking Inpp5b in spermatids and not in other cell types generated by conditional gene targeting, were fully fertile. The abnormalities in mutant sperm function and maturation appear to arise from defects in the functioning of Sertoli and epididymal epithelial cells. Our results directly demonstrate a previously unknown role for phosphoinositides in normal sperm maturation beyond their previously characterized involvement in the acrosome reaction. Inpp5b(-/-) mice provide an excellent model to study the role of Sertoli and epididymal epithelial cells in the differentiation and maturation of sperm.     

The ATP-binding cassette transporter 1 mediates lipid efflux from Sertoli cells and influences male fertility

The liver X receptor/retinoid X receptor (LXR/RXR)-regulated gene ABCA1 effluxes cellular cholesterol and phospholipid to apolipoprotein A1 (apoA1), which is the rate-limiting step in high-density lipoprotein synthesis. The RXR pathway plays a critical role in testicular lipid trafficking, and RXRbeta-deficient male mice are sterile and accumulate lipids in Sertoli cells. Here, we demonstrate that ABCA1 mRNA and protein are abundant in Sertoli cells, whereas germ cells express little ABCA1. LXR/RXR agonists stimulate ABCA1 expression in cultured Sertoli MSC1 and Leydig TM3 cell lines. However, Sertoli TM4 cells lack ABCA1, and TM4 cells or primary Sertoli cells cultured from ABCA1(-/-) mice both fail to efflux cholesterol to apoA1. Expression of exogenous ABCA1 restores apoA1-dependent cholesterol efflux in Sertoli TM4 cells. In vivo, ABCA1-deficient mice exhibit lipid accumulation in Sertoli cells and depletion of normal lipid droplets from Leydig cells by 2 months of age. By 6 months of age, intratesticular testosterone levels and sperm counts are significantly reduced in ABCA1(-/-) mice compared with wild-type (WT) controls. Finally, a 21% decrease (P = 0.01) in fertility was observed between ABCA1(-/-) males compared with WT controls across their reproductive lifespans. These results show that ABCA1 plays an important role in lipid transport in Sertoli cells and influences male fertility.     

Percoll gradient-centrifuged capacitated mouse sperm have increased fertilizing ability and higher contents of sulfogalactosylglycerolipid and docosahexaenoic acid-containing phosphatidylcholine compared to washed capacitated mouse sperm

Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.     

Functional analysis of sperm from c-mos(-/-) mice

The c-mos protooncogene, which is expressed predominantly in male and female germ cells, is crucial for normal oocyte meiosis and female fertility in mice. Inactivation of c-mos results in abnormal oocyte development and leads to ovarian cysts and tumors in vivo. In contrast to the severe effects of c-mos ablation in females, targeted inactivation of c-mos has not been reported to affect spermatogenesis in male mice. However, previously reported studies of male c-mos(-/-) mice have been limited to histological analyses of testes and in vivo matings, both of which are relatively insensitive indicators of sperm production and function. Therefore, we assayed sperm function of c-mos(-/-) males under in vitro conditions to determine whether the absence of Mos during development affected sperm production or fertilizing ability. We found no significant differences between the number of sperm collected from c-mos(-/-) and wild type mice. Additionally, sperm from c-mos(-/-) and c-mos(+/+) males performed equally well in assays of in vitro fertilization (IVF) and fertilization-associated events including zona pellucida (ZP) penetration, sperm/egg plasma membrane fusion, and sperm chromatin remodeling. Therefore, we suggest that the function of Mos in spermatogenesis is either not related to the ultimate fertilizing potential of the sperm, or else the absence of Mos is masked by a redundant kinase.     

Role of sperm sulfogalactosylglycerolipid in mouse sperm-zona pellucida binding

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of mammalian male germ cells. Like other sulfoglycolipids, SGG is believed to be involved in cell-cell/extracellular matrix adhesion. Specifically, we investigated whether sperm SGG played a role in sperm-egg interaction. Initially, we produced an affinity-purified, rabbit polyclonal immunoglobulin (Ig) G antibody that specifically recognized SGG (anti-SGG). Indirect immunofluorescence using anti-SGG IgG localized SGG to the convex and concave ridges and the postacrosome of the mouse sperm head. Pretreatment of sperm with anti-SGG IgG/Fab inhibited sperm-zona pellucida (ZP) binding in vitro in a concentration-dependent manner (to a maximum of 62%). This inhibition was observed at the level of primary binding. Sperm treated with anti-SGG IgG underwent the spontaneous and ZP-induced acrosome reaction at the same rate as control sperm treated with preimmune rabbit serum IgG. Fluorescently labeled SGG liposomes were shown to associate specifically with the egg ZP, whereas fluorescently labeled liposomes of galactosylglycerolipid (SGG's parental lipid) and phosphatidylserine (negatively charged like SGG) did not. Furthermore, coincubation of SGG liposomes with sperm and isolated ZP inhibited sperm-ZP binding in a concentration-dependent manner. These results strongly suggest an involvement of sperm SGG in direct binding to the ZP.     

Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.     

Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken

Transplantation of male germ cells into sterilized recipients has been widely used in mammals for conventional breeding and transgenesis purposes. This study presents a workable approach for germ cell transplantation between male chickens. Testicular cells from adult and prepubertal donors were dispersed and transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. In comparison to males transplanted with testicular cell preparations from adult donors, in which the first ejaculates with sperm were recovered about 5 wk after transfer, a substantial interval (about 10 wk) was necessary to obtain ejaculates after the transfer of testicular cells from prepubertal donors. However, in both cases, recipient males produced ejaculates capable of fertilizing ova and producing progeny expressing donor genes.     

Testis-specific cytochrome c-null mice produce functional sperm but undergo early testicular atrophy

Differentiating male germ cells express a testis-specific form of cytochrome c (Cyt c(T)) that is distinct from the cytochrome c expressed in somatic cells (Cyt c(S)). To examine the role of Cyt c(T) in germ cells, we generated mice null for Cyt c(T). Homozygous Cyt c(T)(-/-) pups were statistically underrepresented (21%) but developed normally and were fertile. However, spermatozoa isolated from the cauda epididymis of Cyt c(T)-null animals were less effective in fertilizing oocytes in vitro and contain reduced levels of ATP compared to wild-type sperm. Sperm from Cyt c(T)-null mice contained a greater number of immotile spermatozoa than did samples from control mice, i.e., 53.1% +/- 13.7% versus 33.2% +/- 10.3% (P < 0.0001) for vas deferens sperm and 40.1% +/- 9.6% versus 33.2% +/- 7.5% (P = 0.0104) for epididymal sperm. Cyt c(T)-null mice often exhibit early atrophy of the testes after 4 months of age, losing germ cells as a result of increased apoptosis. However, no difference in the activation of caspase-3, -8, or -9 was detected between the Cyt c(T)(-/-) testes and controls. Our data indicate that the Cyt c(T)-null testes undergo early atrophy equivalent to that which occurs during aging as a consequence of a reduction in oxidative phosphorylation.     

Quantity rather than quality in teratospermic males: a histomorphometric and flow cytometric evaluation of spermatogenesis in the domestic cat (Felis catus)

Teratozoospermia (ejaculation of <40% morphologically normal sperm) commonly occurs within the Felidae, including certain domestic cats, but the cellular and molecular mechanisms that give rise to this phenomenon remain unknown. This study quantified spermatogenesis to identify differential dysfunctions in teratospermic versus normospermic (>60% normal sperm/ejaculate) domestic cats. Sperm used were from electroejaculates and cauda epididymides. Testes from 10 normo- and 10 teratospermic males were obtained by castration and then evaluated by histomorphometry, flow cytometry, and testicular testosterone enzyme immunoassay. Some morphometric traits (tubular diameter, epithelium height, interstitial area, number of Leydig cells, and blood vessels per cross-section) as well as testicular testosterone concentrations were similar between groups, but testicular volume was greater in teratospermic males. Stage frequencies differed also between both cat populations, suggesting possible dysfunctions in spermiation. Quantification of cell populations in most frequent stages revealed more spermatogenic cells and fewer Sertoli cells per tubule cross-section as well as per tissue unit in teratospermic donors. Hence, the ratio of spermatogenic cells per Sertoli cell was elevated in the teratospermic cat. DNA flow cytometry confirmed higher total spermatogenic and meiotic transformations in teratospermic males. In summary, compared with normospermic counterparts, teratospermic cats have a higher sperm output achieved by more sperm-producing tissue, more germ cells per Sertoli cell, and reduced germ cell loss during spermatogenesis. Gains in sperm quantity are produced at the expense of sperm quality.     

Disruption of mouse CD46 causes an accelerated spontaneous acrosome reaction in sperm

Human membrane cofactor protein (MCP, CD46) is a ubiquitously expressed protein known to protect cells from complement attack. Interestingly, when we examined the expression of mouse CD46, which we recently cloned, the message was found only in testis and the protein was found on the inner acrosomal membrane of sperm. In order to elucidate the function of CD46, we produced mice carrying a null mutation in the CD46 gene by using homologous recombination. Despite the absence of CD46, the mice were healthy and both sexes were fertile. However, to our surprise, the fertilizing ability of males appeared to be facilitated by disruption of the CD46 gene, as the average number of pups born from CD46(-/-) males was significantly greater than that of wild-type males. It was also revealed that the incidence of the spontaneous acrosome reaction doubled in CD46(-/-) sperm compared to that in wild-type sperm. It was assumed that this increase caused the heightened fertilizing ability found in CD46(-/-) sperm. These data suggest that CD46 may have some role in regulating sperm acrosome reaction.     

Protein phosphatase 1cgamma is required in germ cells in murine testis

The protein phosphatase 1cgamma (PP1cgamma) gene is required for spermatogenesis. Males homozygous for a null mutation are sterile, and display both germ cell and Sertoli cell defects. As these two cell types are physically and functionally intimately connected in the testis, the question arises as to whether the primary site of PP1cgamma action is in Sertoli cells, germ cells, or both. We generated chimeric males by embryo aggregation to test whether wild type Sertoli cells are capable of rescuing mutant germ cells. To distinguish between the desired XY-XY chimeras and uninformative XX-XY chimeras, we designed an adaptation of the single nucleotide primer extension (SNuPE) assay. None of the XY-XY chimeras sired pups derived from mutant germ cells, indicating that the protein is required in germ cells for production of functional sperm. Analysis of a chimeric testis revealed intermediate phenotypes when compared with PP1cgamma-/- testes, suggestive of cell nonautonomous effects. We conclude that PP1cgamma is required in a cell autonomous fashion in germ cells. There may be an additional cell nonautonomous role played by this gene in testes, possibly mediated by defective signaling between germ cells and Sertoli cells.     

Sertoli cell development and function in an animal model of testicular dysgenesis syndrome

Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.     

Colocalization of sulfogalactosylacylalkylglycerol (SGG) and its binding protein during spermatogenesis and sperm maturation. Topology of SGG defines a new testicular germ cell membrane domain

By use of double-labelling indirect immunofluorescence, we have shown that the major mammalian testicular glycolipid sulfogalactosylacylalkylglycerol (SGG) and a membrane protein, previously shown to bind specifically to SGG in vitro, are colocalized on the surface of rat testicular germ cells during spermatogenesis. SGG is restricted to convoluted membrane domains within these cells. Thus, the binding affinity in vitro is reflected in the cell surface topology. The topological relationship between these two antigens was also studied during epididymal sperm maturation. Whereas these antigens were colocalized in caput spermatozoa (on the middle and principal piece of the tail and on the concave surface of the head), the distribution of the binding protein was altered for cauda sperm in that the convex surface of the sperm head was now strongly labelled. These studies illustrate the dynamic nature of protein-glycolipid interactions during germ cell differentiation.     

Disruption of spermatogenesis in boars sub-clinically infected with Trypanosoma brucei brucei

Data from 14 crossbred (Landreace x Large white) boars aged 10-12 months were used to investigate specific germ cells and to what extent Sertoli cells are prone to sub-clinical infection with strain Y58/98 Trypanosome brucei brucei and effects on spermatogenesis. Boars were divided into three groups, A, B and C of 5, 5 and 4 animals, respectively. Groups A and B were infected intraperitoneally with 2.8x10(6) trypanosomes per animal. Group C consisted of intact controls. At stable sub-clinical trypanosomiasis, boars in groups A and B together with two from the controls were weighed, scrotal circumferences were measured and animals were castrated on days 56 and 84 post infection, respectively. Testes were weighed. A portion of a testis was processed for histomorphometric assessment and another portion was used to determine gonadal sperm reserves by haemocytometry. Crude cells were converted to true cells.Sub-clinical trypanosomiasis was characterised by low live and testes weights, reduced scrotal circumference, scanty parasitemia peaks at long intervals and decreased libido. Histomorphometry of animals infected with T. brucei brucei revealed somniferous tubular distortion, denudation and or degeneration of germ cells and Sertoli cells leading to distortion of spermatogenesis. Spermatids and young primary spermatocytes were most prone to, while Sertoli cells and spermatogonia were least affected by sub-clinical trypanosomiasis. There was evidence of regeneration of germ cells from precursor stem cells, resulting in slightly increased gonodal sperm reserves as the post infection period increased. Infected boars may not attain original fertility levels consequently. It was concluded that boars in tropical regions that harbour endemic disease should be maintained under prophylactic conditions.     

Male germ cell specific sulfogalactoglycerolipid is recognized and degraded by mycoplasmas associated with male infertility

Sulfogalactosylglycerolipid (SGG) is the major mammalian male germ cell glycolipid and has been implicated in sperm/egg binding. Mycoplasma pulmonis, a species of Mollicutes, is associated with male infertility in rodents. Purified SGG incubated in the presence of M. pulmonis was enzymatically degraded by both desulfation and deacylation. Desulfation occurred primarily at alkaline pH, and deacylation also increased with increased pH, indicating that these represent novel enzymatic activities. Digestion was facilitated, but not dependent on, the presence of detergent. Rat spermatozoa exposed to M. pulmonis showed a reduction in SGG content which was particularly marked for cauda (mature) spermatozoa. With the aid of tlc overlay binding procedure, intact M. pulmonis were found to bind specifically to sulfated glycolipids and thus SGG may provide the cell membrane receptor for this organism. The topology of mycoplasma binding to rat sperm was consistent with the known topology of sperm SGG. The reduced binding (and subsequent digestion) of caput spermatozoan SGG correlates with the membrane colocalization of SGG and its endogenous binding protein at this stage. Separation of SGG and its binding protein during epididymal sperm maturation appears to facilitate M. pulmonis binding to and digestion of cauda sperm SGG. The binding and degradation of the sperm SGG by M. pulmonis may play a role in the induction of infertility which follows infection with these organisms by interfering in sperm/egg receptor recognition.     

Distribution and levels of cellular retinol- and cellular retinoic acid-binding protein in various types of rat testis cells

The distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were measured in rat testicular peritubular and Sertoli cells and in isolated rat pachytene spermatocytes and spermatids. Two Sertoli cell preparations, one containing some germ cells and another that had been osmotically shocked to destroy germ cells, were examined. CRBP and CRABP levels were measured by specific and sensitive radioimmunoassays. Testicular peritubular cell cytosol preparations were found to contain high levels of CRBP (1.48 +/- 0.87 microgram CRBP/mg protein) but CRABP could not be detected. The mean CRBP level in Sertoli cell preparations that contained some germ cells was 0.93 +/- 0.24 microgram CRBP/mg protein; this value was similar to the level of 1.11 +/- 0.20 microgram CRBP/mg protein measured for Sertoli cells free of germ cells. The level of CRABP found in Sertoli cell preparations containing germ cells (0.81 +/- 0.32 microgram CRABP/mg protein) was approximately five times greater than was observed in Sertoli cells free of germ cells (0.16 +/- 0.03 microgram CRABP/mg protein). CRBP and CRABP levels in cultured Sertoli cells were not affected by time in culture for up to five days of culture. Pachytene spermatocytes and spermatids were very enriched in CRABP (0.72 +/- 0.26 microgram CRABP/mg protein for spermatocytes and 0.65 +/- 0.21 microgram CRABP/ml protein for spermatids). A search for a high molecular weight retinol-binding protein did not demonstrate the existence of such a protein in Sertoli cell-conditioned medium. In summary, these studies provide quantitative information about the distribution of the cellular retinoid-binding proteins in the cell types that compose the rat testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of acrosome assembly in a male sterile mouse mutant

Blind-sterile (bs) is a new autosomal recessive mutation of the mouse that causes sterility in males and bilenticular cataracts in both sexes. Sterile bs/bs males exhibited normal copulatory behavior, reduced testis weights, and few or no epididymal sperm. The effects of the bs mutation on spermatogenesis were examined by light and electron microscopy. All sperm present were morphologically abnormal with aberrant head shape. Adult bs/bs testes were characterized by germ cell depletion that resulted in profound alterations of the typical germ cell associations. Only 30% of the tubules contained relatively normal germ cell associations while 39% were extensively depleted, showing only Sertoli cells or Sertoli cells and spermatogonia. The most striking effect of the bs mutation on spermiogenesis was the failure of acrosome formation. Disorganized proacrosomic granules were detected up to step 3 of spermiogenesis by both periodic acid-Schiff staining and ultrastructural analysis. In over 3500 spermatids scored past steps 3-4 of spermiogenesis not a single acrosomal cap or fully developed acrosome was detected. Electron microscopy revealed a thickening of the nuclear envelope of elongating spermatids in the region where the acrosome should have been located; however, no acrosome was present. Chromatin condensation and nuclear elongation did occur in these acrosomeless spermatids, suggesting that caudal growth of the acrosome is not a mechanistic factor in these events.     

Phenotype and fertilizing capacity of spermatozoa of chimaeric mice produced from two strains that differ in sperm quality

Spermatozoa of males from the inbred mouse strains, KE (albino) and CBA (agouti), are distinguishable by head shape and differ in quality: CBA spermatozoa show a lower percentage of abnormal heads and higher efficiency of fertilization. Aggregation chimaeras were produced to investigate whether these differences are intrinsic or extrinsic to spermatogenic cells. Among 24 overt chimaeras, 14 were males: 1 was sterile, 8 produced either KE or CBA spermatozoa, as recognized by shape and by progeny testing, and 5 (21%) were germ-line chimaeras. Both the level of abnormal sperm heads and the efficiency of fertilization of CBA and KE spermatozoa produced by chimaeric males (except two subfertile ones) were within the range characteristic for the respective strain. All 5 germ-line chimaeras, irrespective of their coat colour composition, produced about 98% KE and only 2% CBA spermatozoa, which indicated strong selection against CBA germ cells. However, mature CBA spermatozoa showed high competitive ability, because the proportion of agouti progeny (from the CBA component) sired by those males was significantly higher than the proportion of CBA spermatozoa, estimated from vaginal plug preparations after every mating. The fact that this difference was particularly striking for one chimaera with a preponderance of CBA somatic component may suggest some influences extrinsic to spermatogenic cells. We conclude from this study that sperm head shape, the level of sperm abnormalities and fertilizing capacity are determined largely autonomously by genes acting in the germ cells. The internal environment created by foreign somatic cells exerts only minor modifications, unless there has been deterioration beyond the range of 'normality'.