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C Shaha

Publications and source records attributed to C Shaha.

At least 19 recordsLinked to original sources
PubMed1993

Monoclonal antibody against a human sperm protein recognizes multiple epitopes on rabbit and human sperm and blocks sperm function.

A monoclonal antibody was raised against a human sperm protein of apparent molecular size of 40 kDa. Of the 6 hybridoma clones selected for study, one clone (B-12) was chosen for further investigations. The antibody secreted by the clone agglutinated human and rabbit spermatozoa in vitro. The antibody belonged to IgM class. In indirect immunofluorescence studies this antibody reacted to acrosome of living human and rabbit spermatozoa. On fixed sperm of the same species it recognized midpiece and parts of tail along with the acrosome. Mouse, hamster, guinea pig, monkey and rat sperm showed similar localization. Interaction between rabbit sperm and oocyte was inhibited in vitro by this antibody. On western blots of human and rabbit sperm extracts the antibody recognized more than one epitope.

Animals↗
PubMed1992

Amphiphilic and hydrophilic domains of human sperm membrane antigens recognized by immunoinfertile sera.

Selected sera from married couples with immunological infertility were used to identify antigens on hydrophilic and amphiphilic domains of human sperm membrane. Out of eight sera, six recognized proteins from the hydrophilic as well as amphiphilic regions of the sperm membrane. Sera were either reactive to acrosome or to equator and tail of human sperms in indirect immunofluorescence assay.

Acrosome↗
PubMed1991

Characterisation of 24-kD proteins from rat testes using polyclonal sera reactive to human sperm antigens.

A group of antigens of 24-kD Mr from rat testes were characterised biochemically. These antigens were part of a larger molecule of approximately 200 kD. On treatment with disulfide bond reducing agent, the 200-kD molecule was reduced to subunits. Immunoreactivity was confined to a doublet of approximately 24 kD and a single band of approximately 50 kD Mr after the reduction. Glycoprotein in nature, this antigen shared immunoreactive epitopes with a 40-kD antigen on human spermatozoa. Antiserum raised in rabbits against the 24-kD antigen from rat testes reacted with antigens on the acrosome of human spermatozoa. Agglutination of sperm could be induced by the antiserum. The carbohydrate residue could be removed by mannosidase digestion. Chemical deglycosylation studies showed a slight decrease in molecular weight. Immunoreactivity was however not completely lost after chemical deglycosylation. Isoelectric focusing of the antigen identified nine isoelectric species. Two relatively minor species showed immunoreactivity. Acrosome-reacted spermatozoa showed loss of antigens from acrosome.

Acrosome↗
PubMed1990

Induction of infertility in female rats after active immunization with 24 kD antigens from rat testes.

A unique polyvalent antiserum against whole washed human sperm was used previously to identify groups of antigens on spermatozoa. The antiserum, designated as Antiserum I, recognized a 40 kD antigen in human sperm extracts. Antiserum I caused agglutination of human sperm and prevented interaction of mouse sperm and oocytes. This serum also recognized a band of 24 kD in rat testicular cytosol. In the present study this group of 24 kD proteins was used as an antigen preparation to actively immunize female rats. Immunization was carried out with two different adjuvants: nor-muramyl dipeptide and SPLPS (a thyalated derivative of lipopolysaccharide). Both groups of animals showed significant antibody titres as detected by indirect immunofluorescence and by sperm agglutination tests. In both groups over 80% of the animals remained infertile, compared to 13% of the controls. It is concluded that a group of antigens in rat testes recognized by Antiserum I offer promise as candidates for a contraceptive vaccine.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1989

Characterization of a monoclonal antibody against a 24KD antigen from rat testis important for fertility regulation.

Monoclonal antibodies were raised against a 24KD antigen from rat testicular cytosol which was previously shown to produce antibody titres in rats with inhibitory effect on fertility. Of the 20 hybridoma clones selected for study, one clone HS-D5 was finally chosen for characterisation as it proved to be functionally promising. The clone was secreting IgM type of antibody. It produced strong agglutination of human sperm and prevented binding of hamster sperm to hamster oocyte. In localization studies it identified the acrosome of sperm of multiple species. Western blots with rat testicular cytosol and human sperm extracts showed a strong band at around 24KD with HS-D5. On Western blots from two dimensional gels, HS-D5 identified multiple spots at the region of 24KD. The mechanism of action of the antibody seemed to be at the level of interaction of the oocyte and spermatozoa.

Animals↗
PubMed1989

Immunostainable inhibin subunits are in multiple types of testicular cells.

Immunostainable inhibin alpha-subunit has been demonstrated in rat testes in a pattern consistent with localization in Sertoli cells. In the present study the distribution of alpha-subunit immunostaining was compared to those of beta-A- and beta-B-subunits. Immunostaining of alpha-subunit was present in the seminiferous epithelium of fetal, neonatal, pubertal, and adult rats as well as in Sertoli cells in culture. The distribution of inhibin beta-B-subunit immunostaining in this epithelium was consistent with Sertoli cell localization similar to that of the alpha-subunit. The predominant staining with antibodies against the beta-A-subunit was in nuclei of immature germ cells around the periphery of each seminiferous tubule. The most probable localization of this staining was in the nuclei of pachytene and zygotene spermatocytes. Specific immunostaining with beta-A-subunit antiserum was also evident in the seminiferous epithelium adjacent to the tubular lumen. Immunoreactive alpha- and beta-A-subunit staining was present in a Leydig cell line, and beta-A immunoreactivity was present in interstitial cells of neonatal rat testes. After hypophysectomy, inhibin alpha-subunit immunostaining decreased, beta-A-subunit staining did not change, and beta-B-subunit staining increased. We conclude that immunoreactive inhibin subunits are present in multiple cells in the testis and that the amounts of immunostainable subunits in the seminiferous epithelium are differentially regulated.

Animals↗
PubMed1989

Vaccine for control of fertility.

Birth control vaccines constitute a new category of vaccines. Immunization with the objective of selectively blocking a physiological process differs in many respects from immunizing against pathogens. Conceptually, these widen the orbit of therapeutic intervention by immunological methods. Success recorded in making vaccines regulating fertility offers models to regulate any other physiological process in the body. At a practical level, the task of making such vaccines is beset with inherent difficulties and with new challenges. This article, dedicated to Avrion Mitchison, aims to discuss these problems and to record successes wherever achieved.

Family Planning Services↗
PubMed1988

Identification of sperm antigens that regulate fertility.

Polyclonal antisera were generated in rabbits against human sperm extracts. Out of many such antisera, one was selected (antiserum I) because it recognized relatively few antigens on Western blots. Antiserum I identified immunostainable material on the acrosome of human, monkey, rabbit, hamster, rat and mouse sperm. A detailed histochemical study using rat testes showed that the antigen was detectable on early spermatids and sperm, but not on less mature germ cells. Immunohistochemistry at the electron microscope level localized the antigen on the plasma membrane of rat sperm. In Western blots using human sperm extracts, the antibody recognized a major band with an apparent molecular weight (MW) of 40,000 and minor bands at 43,000 and 72,000. With extracts of monkey sperm and rat testicular cytosol, antiserum I recognized antigens of 69,000 and 23-24,000, respectively. Functionally, antiserum I produced strong agglutination of human sperm. It also prevented attachment of mouse sperm to mouse oocytes in vitro and reduced fertility when administered to female mice. These results suggest that antiserum I can be used as a possible reagent for selecting sperm antigens as components of a contraceptive vaccine.

Animals↗
PubMed1988

Localization of immunoreactive testibumin in the testis and epididymis of adult rats.

Testibumin is a glycoprotein previously isolated from the spent media of primary Sertoli cell-enriched cultures prepared from 20-day-old rats. Immunoassayable testibumin is found in the highest concentrations in testis, epididymis and fluids of the male reproductive tract in adult rats. In the present study, light microscopy was used to show that immunostainable testibumin in paraffin sections of rat testis was localized along the base of the seminiferous epithelium and in finger-like projections from the base of the epithelium, corresponding to the position of the Sertoli cells. The immunostaining of Sertoli cells was shown to be specific since either purified testibumin or crude Sertoli cell-enriched culture medium could compete with antibody for binding sites in tissue sections. The observations using light microscopy were confirmed when Sertoli cells were examined by electron microscopy using a pre-embedding immunostaining technique. The epithelium of the epididymis also contained immunoreactive testibumin which was localized in the caput, corpus and cauda. Immunostainable testibumin was also localized in the corpora lutea of the rat ovary and in the epithelium of the uterine endometrium. These observations are consistent with previous reports that immunoreactive testibumin is present in these organs as demonstrated by radio-immunoassays. We conclude that (i) the immunolocalization of testibumin in Sertoli cells adds to a growing list of observations suggesting that it is made in this cell type; (ii) the other sites of testibumin synthesis in the male are uncertain but the ovary and uterus are possible sites in the female; (iii) electron microscopy following pre-embedding immunostaining and epitope selection can be used as an adjunct to conventional immunocytochemistry to localize proteins in Sertoli cells.

Age Factors↗
PubMed1986

The effects of opioid receptor antagonists suggest that testicular opiates regulate Sertoli and Leydig cell function in the neonatal rat.

beta-Endorphin and other peptides derived from proopiomelanocortin are synthesized in testicular Leydig cells. To better understand the possible function of these and other endogenous opioid peptides in the testis, the opioid antagonists naloxone and nalmefene were administered intratesticularly to hemicastrated 5-day-old rats. Both naloxone and nalmefene potentiated testicular hypertrophy induced by unilateral orchidectomy at 11 days of age. Unexpectedly, at least a 100-fold lower dose of nalmefene was required to produce maximal hypertrophy than that previously reported for naloxone. Leydig and Sertoli cell functions were evaluated, respectively, by measurement of basal testosterone production in vitro and rat androgen-binding protein (rABP) in serum. The optimal dose of naloxone for hypertrophy (1 microgram/testis) suppressed testosterone production and had a nonuniform effect on rABP secretion (either had no effect or produced a slight increase). By contrast, the optimal dose of nalmefene for hypertrophy (0.01 microgram/testis) not only suppressed basal testosterone secretion, but also uniformly increased rABP levels in serum. Larger doses of this opioid antagonist, up to 1 microgram/testis, were not as effective on the three parameters measured (hypertrophy, testosterone secretion, and rABP levels). These results suggest that this agent has both antagonistic and agonistic activities in the testis. At the doses that produced optimal effects on hypertrophy, systemic administration of these antagonists produced no effects. The results of these studies suggest that intratesticular opiates exert a suppressive effect on Sertoli cell growth and rABP secretion. In addition, these peptides may modulate testosterone secretion by Leydig cells.

Androgen-Binding Protein↗
PubMed1984

Identification and possible function of pro-opiomelanocortin-derived peptides in the testis.

Using antibodies against peptides derived from different portions of the POMC molecule, immunocytochemical evidence suggests that this precursor and/or the peptides present within it are localized in testicular Leydig cells of at least five species. There is no evidence for the localization of these peptides or their precursor in any other cell type in this organ. Examination of testicular extracts by gel filtration, SDS-PAGE, and RP-HPLC indicate that the testis contains low concentrations of POMC-derived peptides relative to brain. Further analysis indicates that POMC is processed to alpha-MSH and beta-endorphin similar to its processing in intermediate pituitary lobe and brain. The relative mobilities of immunoreactive alpha-MSH and beta-endorphin on RP-HPLC columns indicate that they are in the unacetylated state as in brain and in contrast to the acetylated forms in the intermediate pituitary lobe. The potential for Leydig cells to synthesize POMC and its peptides was suggested by the demonstration of POMC-like mRNA in total testis and Leydig cell cultures. The size of the POMC-like mRNA is approximately 150 base pairs shorter than anterior or intermediate pituitary POMC mRNA. POMC-like mRNA activity has also been localized to Leydig cells in sections of testes using in situ hybridization. Immunostainable beta-endorphin and other POMC-derived peptides are present in testicular Leydig cells during fetal life and following puberty at times when testosterone secretion is maximal. The accumulation of immunostainable POMC-derived peptides in Leydig cells is dramatically increased by LH and hCG. A variety of observations suggests that testicular cells can respond to POMC-derived peptides. ACTH and the MSHs stimulate growth and cAMP accumulation in Sertoli cells. By contrast, studies using antagonists suggested that beta-endorphin and/or another testicular opioid inhibit Sertoli cell proliferation and ABP secretion. These observations are consistent with the postulate that different portions of the POMC molecule may have opposite effects on Sertoli cell function and suggest a mechanism by which Leydig cells could modulate Sertoli cell activity. Intratesticular administration of opiate antagonists inhibits testosterone secretion both in vivo and in vitro. These observations suggest that Leydig cell-derived beta-endorphin may facilitate testosterone secretion either directly or indirectly. The finding of POMC and its derivative peptides in testis, ovary, adrenal, and placenta suggests that all steroid hormone-secreting organs in mammals may utilize this peptidergic system.

Adrenocorticotropic Hormone↗
PubMed1984

The ontogeny of immunoreactive beta-endorphin in fetal, neonatal, and pubertal testes from mouse and hamster.

UNLABELLED: Derivatives of proopiomelanocortin (POMC), physicochemically similar to beta-endorphin and desacetyl alpha MSH, have been identified in adult testes, where these peptides were localized to Leydig cells. In the present study, the presence of immunostainable derivatives of POMC was established in fetal, neonatal, and pubertal testes with the unlabeled antibody peroxidase-antiperoxidase method. Specificity of staining was established by absorption of primary antisera with excess antigen. In the mouse, immunoreactive beta-endorphin was detectable in a few primitive interstitial cells on day 14 of gestation, the day after testicular differentiation. Thereafter, the number of immunostainable cells progressively increased throughout fetal life, so that at birth, they comprised 55% of the total interstitial cells. After birth, the number of immunostaining cells declined, so that they were only 12% of interstitial cells by 5 days of age. After 10 days of age, the number of immunopositive cells progressively increased, and by 40 days, interstitial cells showed intense staining comparable to that in adult mice. At 10 days of age, when the number of immunostainable cells was low, hCG treatment increased both the number and staining intensity of beta-endorphin-positive cells to those seen in adult testes. Antibodies directed against gamma MSH, a peptide within the N-terminal segment of POMC, also produced specific staining of fetal and adult interstitial cells in the mouse. In the hamster, the pattern of staining with anti-beta-endorphin in fetal, neonatal, and pubertal interstitial cells was similar to that observed in mice; the number and staining intensity of immunostainable cells increased during fetal life, declined after birth, and rose again at puberty. IN CONCLUSION: 1) the number and staining intensity of immunostainable interstitial cells have two peaks in mouse and hamster, at birth and after puberty; 2) the number and staining intensity of mouse interstitial cells can be increased by hCG; and 3) the development of immunostainable beta-endorphin activity correlates with the previously reported spontaneous and hCG-induced maturation of morphology and enzyme activities of Leydig cells.

Animals↗
PubMed1984

Demonstration of immunoreactive beta-endorphin- and gamma 3-melanocyte-stimulating hormone-related peptides in the ovaries of neonatal, cyclic, and pregnant mice.

Antisera against proopiomelanocortin (POMC)-derived peptides have previously been employed to demonstrate immunostainable materials in the male reproductive tract and in the corpus luteum of rat ovary. The present study was designed to determine how the distribution of such stainable materials varies in mouse ovary as a function of the reproductive status of the animal. Peptide-like activities were localized with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique in ovaries removed from mice during fetal and neonatal development, during different stages of estrous cycle, and during pregnancy, with antisera against beta-endorphin, gamma 3MSH, and an extended N-terminal portion of POMC (16 K). beta-endorphin-like activity was also quantified in ovarian extracts by RIA. Immunostainable beta-endorphin, gamma 3MSH, and 16 K fragment-like activities were present in ovaries of pregnant and normally cycling (but not immature) mice. Intense staining was found predominantly in the corpora lutea. Less intense staining was observed in the interstitium and in the following parts of large follicles: parietal granulosa, corona radiata, and cumulus oophorus. When neonatal mice were injected with hCG, immunostainable beta-endorphin-like material in the ovarian interstitial area increased. Treatment with PMSG increased staining in both secondary follicles and the interstitium. Immunoassayable beta-endorphin-like activity was twice as high (per g wet wt) at pregnancy as during the cycle. We conclude that peptides similar or identical to POMC and/or its components are present in ovarian cells and that the concentration of such material appears to be regulated by gonadotropins.

Aging↗
PubMed1984

Do testicular opiates regulate Leydig cell function?

beta-Endorphin is believed to be synthesized in testicular Leydig cells. To gain more information about the role of this and other endogenous opioid peptides in the testis, opiate antagonists (naloxone and nalmefene, 100 micrograms/testis) were administered intratesticularly to hemicastrated adult rats. Leydig cell function was evaluated by measurement of serum testosterone and testosterone production in vitro. Estimation of androgen binding protein (rABP) was used as an index of Sertoli cell function. Serum testosterone was reduced significantly by intratesticular administration of naloxone and nalmefene in treated animals. Systemic administration of these antagonists had no effect at the doses used. Testes from treated animals incubated in vitro with or without hCG produced significantly less testosterone than vehicle-treated control testes. Hemicastration reduced rABP synthesis and secretion; however, treatment with opiate antagonists did not alter the amount of this protein in the serum or epididymides of these rats. These observations suggest that endogenous testicular opiates modulate testosterone secretion by Leydig cells.

Androgen-Binding Protein↗
PubMed1984

A species difference between hamster and rat in the effect of oestrogens on growth of large preantral follicles.

Intact or hypophysectomized 23-day-old hamsters and rats were injected s.c. with 2 mg diethylstilboestrol (DES) or 1 mg oestradiol cyclopentylpropionate (OECP) on Days 23-25 and killed on Day 26. Although serum oestradiol was elevated to the same high levels by OECP, ovarian and uterine weights were increased in the rat by OECP or DES whereas only the uterus responded in the hamster. This correlated with the ability of the oestrogens to increase significantly the number of large preantral and antral follicles in the intact rat but only the number of follicles with 2-3 layers of granulosa cells in the immature hamster. Qualitative study revealed that DES and OECP increased the number of large preantral follicles in the adult hypophysectomized rat but were ineffective in the adult hamster. It is concluded that for the immature and adult hamster oestrogens do not play a major role in the recruitment of large preantral follicles.

Animals↗