A new approach to immunological sexing of sperm.

A non-invasive, immunological method for sexing mammalian sperm would be of benefit to agricultural industries. This paper presents a new approach, based on the hypothesis that sex-specific proteins (SSPs) are evolutionarily more highly conserved than non-SSPs. Antibodies to non-SSPs were raised and used in an affinity procedure to remove non-SSPs and enrich for SSPs. Thereafter, using column chromatography, purified SSPs were obtained. Sex-specific antibodies (SSAbs) raised against these SSPs appear to bind to sex-chromosome-specific proteins (SCSPs) on the sperm membrane and make possible a sperm-sexing procedure. Antibodies to SCSPs were raised and used to identify putative SCSPs by affinity chromatography. The preliminary results presented here suggest that a viable immunological sperm sexing procedure can be developed.

Extracellular matrix abnormalities in testis and epididymis of XXSxr ("sex-reversed") mice.

Sex-reversed (Sxr) is a duplication of the sex-determining region of the Y chromosome, which gets transposed to a paternal X chromosome. Chromosomally female (XX) zygotes that receive this XSxr chromosome develop as apparent males. Previous work on XXSxr mice (called pseudomales) showed extracellular matrix (ECM) ultrastructural abnormalities in the epididymis and testis. This study examined the biochemical nature of these abnormalities. More hydroxyproline (an indicator of collagen) was noted in the pseudomale testis and epididymis compared to normal male tissues. Western blot analysis showed increased collagen IV in the pseudomale testis and epididymis. In both the hydroxyproline and collagen IV studies, the epididymis was found to contain higher levels of these substances than the testis for both genotypes. There also appeared to be increased messenger RNA for tissue inhibitor of metalloproteinases (Timp), a regulator of collagen, in the pseudomale testis. Data from these studies seem to indicate that the XXSxr genotype influences ECM deposition and/or turnover and exerts a direct genetic influence on the development of the testis and epididymis. According to the existing paradigm of mammalian sexual development, the epididymis is expected to be normal in the presence of adequate androgenization and independent of chromosomal and genetic sex. The results presented here differ from what would be predicted by this paradigm.

Transcription of circular and noncircular forms of Sry in mouse testes.

Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28-day-old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14-day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells--RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross-hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3' region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT+) and RT- reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre- and postmeiotic germ cells and in somatic cells of the testes.

Zfy is transcribed in the normal mouse epididymis and in the XXSxr ("sex reversed") testis.

The presence of the mutation Sex reversed (Sxr), a copy of a Y-chromosomal segment that gets transferred to an X chromosome, causes the resulting XXSxr mice to develop as apparent males. However, several features of male sexual development are abnormal in these animals. The testes are small and aspermatogenic, and the epididymides lack the initial segment. Testes and epididymides show abnormalities of extracellular matrix. In this study we examined transcription of the conserved Y chromosomal gene Zfy, which has an X-chromosomal homologue (Zfx). Northern blotting showed Zfy to be expressed in the testes of XXSxr animals, except for those that carry the coat-marker gene Tabby (Ta), despite the lack of germ cells in XXSxr mice. Reverse transcription polymerase chain reaction (RT-PCR) studies detected Zfy in mRNA in testes even when Ta was present. RT-PCR also demonstrated Zfy transcription in epididymides of normal males, though not in XXSxr mice. Previous authors reported an absence of Zfy transcription in XXSxr testes; Zfy transcription in normal testes has been ascribed to germ cells. Our observation indicates that this idea requires re-evaluation. The occurrence of Zfy transcription in the normal epididymis is similarly a novel finding that may help explain those aspects of epididymal development that occur in the absence of androgen.

Penoscrotal transposition: a case report and review.

We report on a boy born with complete penoscrotal transposition, normal scrotum, twisted penile shaft with hypoplastic penile urethra, meatal stenosis, normal bladder, and bilateral cystic dysplastic kidneys. The patient died of renal failure at 2.5 months. This is the 13th reported case of complete penoscrotal transposition with normal scrotum. The possible pathogenesis is discussed and the literature is reviewed.

Histological studies on eyelid opening in normal male mice and hemizygotes for the mutant gene Tabby (Ta) with and without epidermal growth factor treatment.

Previous work has shown that mice hemizygous or homozygous for the mutant gene Tabby have delayed eyelid opening, as compared to unaffected, wildtype littermate controls; exogenous treatment with epidermal growth factor reverses this delay. We performed histological studies to explore the mechanisms of action of the Tabby gene and of epidermal growth factor in these processes. These show that eyelid opening is associated with keratinization of the fusion junction and conjunctival sac formation. Both these processes occur earlier in normal male mice (days 4 and 7 respectively) than in Tabby hemizygotes (days 7 and 10, respectively). After epidermal growth factor injection, keratinization and conjunctival sac formation are both observed on postnatal day 1 in all control and mutant pups. Thus epidermal growth factor appears to accelerate eyelid opening by stimulating these morphological processes and the Tabby gene appears to delay eyelid opening by impairing them. It is possible that deficiency of epidermal growth factor at the tissue level may be involved in the development of some of the traits seen in Tabby mutants. In addition to analysing the effects of the Tabby gene and of epidermal growth factor on eyelid opening in the mouse, this study appears to be the first detailed histological description of normal eyelid opening. The findings have potential clinical significance; firstly, because the Tabby gene shows genetic homology to the human gene for hypohidrotic ectodermal dysplasia, and disturbed eyelid opening is a trait of some forms of human ectodermal dysplasia, and secondly, because the gene for epidermal growth factor receptor is an oncogene.

Development of the normal XY male and sex-reversed XXSxr pseudomale mouse epididymis.

XXSxr pseudomale mice (chromosomally XX animals "sex-reversed" by the Sxr factor) develop testes and produce sufficient androgens for masculinization as assessed at the macroscopic level. However, adult XXSxr pseudomales lack the epididymal initial segment (I.S.). In this study prenatal and postnatal epididymal development was examined histologically and biochemically, and it was found that XXSxr pseudomales are indistinguishable from normal XY males up to day 21 of postnatal life. By 25 days postnatally, before the onset of the pubertal androgen surge, the I.S. precursor is evident in normal animals but absent in XXSxr mutants. No major abnormalities were seen in other segments of the XXSxr epididymis. Our data suggest that androgen levels in testis and epididymis are not higher in normal XY males than in XXSxr pseudomale mice of the same age. Inadequate availability of androgens at the target site is unlikely to be the cause of the epididymal abnormality in XXSxr pseudomale mice.

Induction of sweat glands by epidermal growth factor in murine X-linked anhidrotic ectodermal dysplasia.

Tabby (Ta), a murine X-linked mutant gene, produces a syndrome of ectodermal dysplasia including anhidrosis (absence of sweat glands). Development of sweat glands is related to that of dermal ridges (dermatoglyphics) and abnormal ridges may be associated with absence of sweat glands in the human syndrome of hypohidrotic ectodermal dysplasia (HED). We have found that dermal ridges occur in normal mice but are lacking in Ta mutants. Previously we showed that epidermal growth factor (EGF) reverses delayed eyelid opening and incisor eruption in Ta mice. We now report that EGF induces development of dermal ridges and functional sweat glands in Ta/Y hemizygotes, indicating a role in mammalian morphogenesis. Ta seems to be genetically homologous to human X-linked HED, as Ta maps close to loci homologous to linkage markers of HED and the two syndromes share many traits, including absence of all or most sweat glands. Absence of these glands causes hyperpyrexia, a clinical emergency in infants with HED; reversal of the trait in the mouse homologue of the disease indicates that an important genetically determined congenital defect in humans may become treatable.

Glucocorticoid-induced polycystic kidney disease--a threshold trait.

Administration of hydrocortisone acetate (250 mg/kg) to newborn mice caused polycystic kidney disease (PKD) of varying proportions in each of 18 different inbred strains; none of the injected controls were affected. All kidneys were histologically examined and scored for degree of cyst formation using a semi-continuous (0 to 4+) grading scheme. Results suggested that this condition is a multifactorial threshold trait. For each strain, estimates of the mean and standard deviation of normally distributed liability were determined by maximum likelihood methods. Concomitant analyses showed: 1) a significant environmental effect related to drug source; 2) a variation in thresholds ranging from 0.94 (N = 46) for the B10.M strain to -0.71 (N = 297) for the C57B1/6J strain; and 3) three groups of strains with different susceptibility to PKD. These results are consistent with a multifactorial basis for susceptibility to PKD. Quantitative analysis of thresholds and liability distributions reveals that genetic, environmental and random elements all contribute to the expression and extent of the cystic trait.

Effect of the X-linked gene Tabby (Ta) on eyelid opening and incisor eruption in neonatal mice is opposite to that of epidermal growth factor.

Studies on eyelid opening and incisor eruption in 216 neonatal Tabby (Ta)-bearing mice and wildtype controls (35 Ta/Y, 62 + /Y, 30 Ta/Ta, 57 Ta/+ and 32 +/+) showed that in animals hemizygous and homozygous for Ta, the timing of eyelid opening and incisor eruption was significantly delayed (P less than 0.05). It was also observed that once open, the eyes of mutant pups do not remain open for long but soon close again for several days before reopening. An iterative eyes open-eyes closed process seems to continue beyond puberty. Studies in 25 epidermal growth factor (EGF)-treated mutants and 23 saline-treated controls showed that neonatal EGF injections (4 micrograms g-1 body weight per day) reversed the delayed timing of eyelid opening and incisor eruption in hemizygote and homozygote Tabby mice. However, both mutant and wildtype EGF-treated mice also showed the eyes open-eyes closed cycle, whereas untreated nonmutant mice did not. Because Tabby appears to be genetically homologous to the gene for human X-linked hypohidrotic ectodermal dysplasia, these results may have potential clinical significance. The eyes open-eyes closed cycle may involve cycling levels of EGF receptor; since the gene for this receptor shows homology with an oncogene, this system may be useful in studies on genetic control of oncogene function.

Ultrastructural abnormalities of epididymal tissues in XXSxr pseudomale (sex-reversed) mice.

Sex reversed (Sxr), a duplication of the Y chromosomal testis-determining factor in mice, causes testis development in XXSxr animals. No effects of Sxr on nongonadal organs are expected. However, we have previously shown that the epididymis of XXSxr pseudomale (sex-reversed) mice lacks the Initial Segment. In the present study we examined the ultrastructure of the head of the epididymis of adult and 21-day old XXSxr pseudomale mice. Epithelial cells of both adult and juvenile XXSxr animals contain numerous vesicles, some within mitochondria. The basal lamina is thickened and infolded. The periepithelial layer is abnormally thick, with distorted smooth muscle cells and fibrocytes that also contain lysosomelike vesicles, often in mitochondria, and excessively wide intercellular spaces. Normal collagen fibrils are infrequent; they are in part replaced by wisps of nondiscrete material, possibly immature collagen. Sxr is known to affect spermatogenesis and Sertoli cells. The abnormal conjugation of sex-determining genes in XXSxr appears also to subvert mesenchymal-epithelial development in both epididymis and testis. We believe the most likely explanation of our data is that the XXSxr genotype is not testis specific but also influences the epididymis directly.

Epidermal growth factor (EGF) affects sulphydryl and disulphide levels in cultured mouse skin: possible relationship between effects of EGF and of the tabby gene on thiols.

Studies from our laboratory have previously shown that the syndrome produced in the mouse by the X-linked gene tabby (Ta) has many features in common with human X-linked hypohidrotic ectodermal dysplasia. We have also demonstrated that tabby has abnormally elevated epidermal sulphydryl (SH): disulphide (SS) ratios, in common with an autosomal form of ectodermal dysplasia. The organs and tissues affected in many of the traits of these syndromes are targets of epidermal growth factor (EGF) and we have shown that the EGF-producing cells are deficient in tabby. In the present study we examined whether EGF affects SH and SS levels in normal mouse skin in tissue culture, and we report here that it does. EGF at a concentration of 25 ng/mL tissue culture medium lowers SH levels as compared with controls (0 ng/mL) in the epidermal layers examined (stratum malpighii of the tail and stratum malpighii and stratum corneum of flank skin). In general, other concentrations of EGF increase epidermal SH levels, although very high doses also reduce them. EGF at 25 ng/mL also lowers total SH + SS concentrations in the epidermal layers. Fetuses hemizygous for the Ta gene appear to have higher total SH + SS epidermal concentrations than their wild-type control littermates. These data, taken together with some of our previous findings, suggest the possibility that a relationship may exist between Ta, EGF, and thiol concentrations. Further study is required to elucidate this relationship.

Polycystic kidney and liver disease and corticosterone changes in the cpk mouse.

Mice homozygous for the mutation cpk develop a lethal infantile polycystic kidney disease (PKD) and have abnormal elevations of corticosterone in the postnatal period. We examined the development of these elevated glucocorticoid levels in early life. The histological progression of a cystic lesion of the biliary ducts in older heterozygotes was also studied. Normal mice had low (less than 85 nmol/liter) or undetectable (less than 36 nmol/liter) levels from the sixth to twelfth day of life. Corticosterone levels were significantly higher in all homozygote (cpk/cpk) and some of the normal sibs, who were presumed to be heterozygote (cpk/+) mice compared to age-matched controls from day seven to 12. Corticosterone levels were similar from the fifteenth day. A significant proportion of adult obligate heterozygotes from breeding pairs were found to have focal areas of cystic dilatation of the biliary ducts, in the absence of any renal abnormality. The incidence of this lesion increased with age (10% at 150 days, 23% at 300 days, 29% at 450 days, 65% at greater than 450 days of age). The livers of homozygote mice dying in infancy, and control adult mice of the C57BL/6J background strain were free of cysts. The finding of hepatic cysts is reminiscent of adult type PKD. The cpk model thus supports the concept that the different forms of PKD may represent varying expression of a similar gene complex.

Beta-glucuronidase activity is present in the microscopic epididymis of the Tfm/Y mouse.

The sex-linked recessive gene Tfm in the mouse produces a condition of testicular feminization (androgen insensitivity syndrome, AIS) in hemizygotes, comparable to the condition of the same name in humans. The murine mutant was originally believed to have no derivatives of the mesonephric duct system (MDS), and this absence was ascribed to dependence of these derivatives on androgens for survival. However, microscopical epididymides, retia testes, and vasa deferentia were identified in these animals in our laboratory. These micro-organs may play a role in meiosis induction in Tfm/Y animals. The present study was designed to determine whether survival of these organs is due to retention of an ability to respond to androgens, or whether they are unique amongst MDS derivatives in being independent of androgens. Previous studies in our laboratory demonstrated that the enzyme beta-glucuronidase (beta G) is androgen sensitive in the epididymis of the normal mouse. In the present investigation we used this enzyme as a marker to study androgen sensitivity in the microscopical epididymides of Tfm/Y hemizygotes and in the epididymides of control +/Y litter-mate brothers. Both mutant and control animals were studied with and without exogenous androgen stimulation. Tfm/Y hemizygotes demonstrated low levels of diffuse, cytoplasmic beta G activity that appears to be unresponsive to exogenous androgen stimulation. In light of our previous studies, this distribution of beta G reaction products suggests some degree of androgen sensitivity. The survival of these micro-organs and their partial androgen sensitivity may be related to the role of the MDS in inducing meiosis.

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.

Anhidrosis and absence of sweat glands in mice hemizygous for the Tabby gene: supportive evidence for the hypothesis of homology between Tabby and human anhidrotic (hypohidrotic) ectodermal dysplasia (Christ-Siemens-Touraine syndrome).

I have suggested that the X-linked gene Tabby (Ta) and its autosomal mimics in the mouse may be homologous with the genes for sex-linked anhidrotic (hypohidrotic) ectodermal dysplasia (Christ-Siemens-Touraine syndrome, CST) and its apparent autosomal mimics in the human. In the present study, I examined whether anhidrosis, a cardinal feature of CST, is present in the putative mouse sex-linked model, Tabby. The results demonstrate that whereas normal mice perspire on the volar and plantar surfaces of their paws, hemizygous Ta/Y male mice show anhidrosis and absence of sweat glands, as do human hemizygous male sufferers of CST. This result is strongly supportive of the hypothesis that Ta is homologous to the gene for CST.

Androgen levels and androgenization in sex-reversed (XXSxr pseudomale) mouse: absence of initial segment of epididymis is independent of androgens.

The XXSxr pseudomale ("sex-reversed") mouse has an apparently normal male phenotype. This is in accordance with Jost's Principle that, in an animal with androgen-producing testes, phenotype will be fully masculinized, irrespective of chromosomal sex. However, the epididymis of this mutant lacks the Initial Segment. Several alternative explanations were proposed. In the present study we examined the primary of these, viz, androgen insufficiency. We studied androgen levels in serum and tissues of the mutant, and we investigated whether injection of exogenous androgens could induce normal development. The XXSxr pseudomale is not deficient in androgens, nor do exogenous additional androgens induce development of the Initial Segment. It would appear that the abnormal genotype acts on the organ phenotype without intervention of androgens.