Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli.

An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli. Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 M) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding. Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both alpha-helical and beta-sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization.     

Molecular cloning and expression in Escherichia coli of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for an immunocontraceptive vaccine. The efficacy of such a vaccine has to be evaluated in nonhuman primates, thus necessitating the characterization of their ZP glycoproteins. A bonnet monkey (Macaca radiata) ovarian cDNA λgt11 library was screened for ZP2 (bZP2) using full-length human ZP2 cDNA as a probe. Two identical full-length clones with an open reading frame of 2235 nt encoding a polypeptide of 745 aa residues were isolated. The deduced aa sequence of bZP2 revealed high sequence identity (94.2%) with human ZP2. The bZP2 cDNA (115–1914 nt, 1.8 kb), excluding sequences coding for N-terminal signal sequence and C-terminal transmembranelike domain, was PCR amplified and Sac1-Sal1 restricted fragment cloned in frame downstream of the T5 promoter under the lac operator control in a pQE-30 vector. Recombinant bZP2 (r-bZP2) was expressed as a polyhistidine fusion protein in Escherichia coli strain M15 [pREP4]. Immunoblot with rabbit polyclonal antibodies against bZP2 synthetic peptide (corresponding to aa residues 429–444; K₄₃₄ replaced by R and I₄₃₆ by V) revealed a major band of 68 kDa. Immunization of male rabbits with the r-bZP2 protein purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with r-bZP2 in ELISA as well as with native protein as revealed by positive fluorescence of ZP of bonnet monkey ovary. The availability of r-bZP2 and its aa sequence will help in the development and evaluation of a contraceptive vaccine based on ZP2. Mol. Reprod. Dev. 50:229–239, 1998. © 1998 Wiley-Liss, Inc.     

Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding

To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.     

Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis

Zona pellucida glycoprotein-3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223-348 [bmZP3(223-348)] has been cloned and expressed using baculovirus expression system. SDS-PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of approximately 25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3(223-348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome-reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r-bmZP3(223-348) and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3(23-348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3(23-348) to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3(23-348), r-bmZP3(223-348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r-bmZP3(23-348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3-mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization.     

Growth and reproductive parameters of bonnet monkey (Macaca radiata)

The present paper summarizes some of the important biological and physiological data recorded over a 30-year period on the biology of bonnet monkeys in captivity. Data on sexual maturity, menstrual cyclicity, general behaviour, endocrine profile, reproductive physiology, gestation, parturition, postpartum amenorrhoea in the female, and sexual maturity, hormone profile, and seasonal variation in sperm count of the male monkeys are presented. In addition to the biological values, weights of selected organs, vertebral and dental pattern are also presented. Menarche occurred at an age of 36±4 months and the first conception in the colony occurred at an age of 54±4 months. The average menstrual cycle length was 28±4.3 days. Majority of monkeys did not cycle regularly during March–June during which the temperature reached a peak. The pregnancy index of the colony was 80% with controlled breeding. The gestation period was 166±5 days with 6–7 months postpartum amenorrhoea. Males attained sexual maturity by the age of 6–7 years and exhibited the characteristic nocturnal surge of serum testosterone at this age and sperm concentration ranged from 116–799 millions/ejaculate.     

Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity

An internal fragment (978 bp) corresponding to the bonnet monkey (Macaca radiata) ZP3, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by PCR from a full-length cDNA clone. The amplified Bam HI and Sacl restricted fragment was cloned in frama downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant ZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strains SG13009[pREP4] and BL-21(DE3). Immunoblot with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β—a homologue of bonnet ZP3, and cross-reactive with bonnet zona pellucida) revealed a predominant band of 50 kDa besides degraded fragments. Optimum expression of r-ZP3 was observed at 0.5 mM IPTG. Antisera generated in monkeys against synthetic peptides from the N-(23–45 aa residues) and C-(300–322 and 324–347 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequence reacted with the r-ZP3 protein in ELISA. The r-ZP3 expressed in SG13009[pREP4] was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toxoid (DT). Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate generated antibodies reactive with r-ZP3 in ELISA. Rabbit r-ZP3 antiserum reacted with porcine ZP3β and bonnet r-ZP3 but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The availability of r-ZP3 will further help in evaluating its efficacy for fertility regulation and understanding the autoimmune oophoritis associated with ZP3 immunization in nonhuman primates. Mol. Reprod. Dev. 47:140–147, 1997. © 1997 Wiley-Liss, Inc.     

Studies on ovarian quiescence in the lactating bonnet monkey (Macaca radiata)

Lactating bonnet monkeys were used as a model to understand the mechanism of ovarian quiescence during lactation. The ovary of the bonnet monkey in the 3rd month of lactation responds well to exogenous pregnant mare serum gonadotropin stimulation with serum estrogen values reaching maximal levels by day 3 of the gonadotropin injection. The adminstration of ovine prolactin to such monkeys significantly inhibited the ovarian responsiveness to exogenous gonadotropin. The responsiveness of the pituitary of the lactating monkey (in the 3rd month of lactation) to luteinizing hormone releasing hormone injection was suppressed and supplementation with exogenous prolactin further accentuating this effect. The relative ability of chlorpromazine given intravenously/intramuscularly/intranasally to enhance endogenous prolactin levels was assessed. During the first 5 months of lactation when the basal prolactin levels were high, the luteinizing hormone levels were low. As the suckling stimulus reduces and prolactin levels fall, luteinizing hormone levels increase, the first post-parturient mensus occurring by 218 ± 4 days. This event was postponed by 3 months on increasing endogenous prolactin levels by administering chlorpromazine (250 μg/day by intranasal mode) over a 5 day period every month starting from the 3rd month of lactation.     

Estrogen modulation of riboflavin carrier protein in the bonnet monkey (Macaca radiata)

Circulatory concentrations of riboflavin carrier protein (RCP) were quantitated in bonnet macaques by employing a heterologous radioimmunoassay involving 125I-labelled chicken RCP and its antiserum. The levels of monkey RCP in the serum seem to be governed by the estrogenic status of the animals. An increase in concentration of serum estradiol in the adult females during the menstrual cycle and early pregnancy could be correlated with enhanced serum RCP levels. Estadiol-17 beta administered to both immature female and male monkeys, specifically brought about elevated levels of RCP with a slower time course of response in males than in females. These results could be a reflection of a more rapid decline of both circulatory estrogen and RCP concentrations in male serum. Repeated administration of estradiol-17 beta to male animals led to prolonged elevated levels of RCP following estrogen administration. Thus, it would appear that the evolutionary conservation of RCPs from the aves to the primates encompasses not only their physicochemical similarities but also extends to the estrogenic modulation of their elaboration.     

Estradiol levels near the time of ovulation in the bonnet monkey (Macaca radiata)

Plasma estradiol-17beta and total progestins were determined to delineate the relationship between preovulatory estradiol-17beta peak and ovulation in the bonnel monkey (Macaca radiata). 6 monkeys were studied for 15 menstrual cycles. In subsequent cycles, serial laparotomy was performed in 5 of the 6 monkeys to correlate ovarian morphology to plasma estradiol-17beta. In 11 of the 15 cycles, estradiol-17beta peaks were 3- to 7-fold above baseline levels near the time of expected ovulation (Cycle Days 7-12). Plasma progestin rose significantly from follicular phase levels of .5 ng/ml to 2.6 ng/ml the day of the estradiol-17beta peak with peak levels of 4.5 ng/ml on the following day. Ovarian morphology in 4 of the 5 observed by laparotomy demonstrated ovulation within 48 hours following an estradiol-17beta peak of approximately 300 pb/ml.     

Antifertility effect of tamoxifen as tested in the female bonnet monkey (Macaca radiata)

The administration of a potent antiestrogen, tamoxifen at a dose of 3 mg/kg body weight/day orally post-coitally to cycling mated bonnet monkeys(Macaca radiata) from days 18–30 of cycle resulted in inhibition of establishment of pregnancy in 9 out of 10 monkeys. Tamoxifen effect was not due to interference with luteal function. The effect was specific to tamoxifen as exogenously administered progesterone could not reverse it. In addition to suggesting a role for estrogen in maintenance of early pregnancy in the primate the present study could be a prelude to the development of an effective post-ovulatory approach for regulation of fertility in the human female     

Diurnal variations of serum testosterone levels in the male bonnet monkey (Macaca radiata)

Diurnal variations in serum testosterone levels were studied in six adult maleMacaca radiata. Though the testosterone levels fluctuated considerably during the 24-hr period, a distinctive diurnal pattern characterized by lower concentrations during day hours and higher concentrations during night hours was observed.     

Temporomandibular malformations in the bonnet monkey (Macaca radiata) fetus following maternal ingestion of thalidomide

Structural abnormalities of the temporomandibular region were seen and described in the fetal bonnet monkey (Macaca radiata) after maternal ingestion of thalidomide. A single oral dose of 10 mg/kg of thalidomide was given on day 24, 25, 26, 27, 28 or 29 or 30 mg/kg on day 25 or 28 of gestation to 25 pregnant monkeys (day of mating = 0). The skeletons, processed and stained with alizarin red S, were examined for gross changes in configuration of craniofacial bones. Temporomandibular defects, in varying severity, were seen in four 70-day-old and seven 100-day-old fetuses associated with a single treatment between day 24 and day 28. The highest risk appeared to occur following treatment on day 25 (4/5) and the lowest on day 28 (2/8). Characteristic expression of the defects included 1) temporo-zygomatic hyperplasia involving the articular tubercle and postglenoid process and 2) mandibular hypoplasia, primarily of the condyles and the angular and coronoid processes. Micrognathia, apertognathia, and partial extraarticular ankylosis were seen. Although the sensitivity periods of the temporomandibular and ear regions seem to overlap and the embryonic origins are similar, the defects of each region appeared to be independent. Although the ear demonstrated a definite temporal relationship characterized by a decrease in severity in the expression of the ear malformations with respect to increased gestational age at the time of thalidomide insult, the temporomandibular region failed to exhibit a specific pattern. Although the temporo-zygomatic hyperplasia was dissimilar to any reported human condition, the mandibular changes were similar.     

Malignant fibrous histiocytoma in a bonnet macaque (Macaca radiata)

Spontaneous sarcomas, not associated with an underlying disease, appear to be relatively rare in nonhuman primates. Since 1970, there have been few reported cases of naturally-occurring sarcomas of any kind in these species. A malignant histiocytoma and malignant fibrous histiocytoma have been described in a rhesus macaque and baboon, respectively. A malignant fibrous histiocytoma is defined as a sarcoma of varied pattern consisting of a mixture of histiocytic and fibroblastic elements. It is thought that the two cells types arise from a common precursor or that the fibroblastic elements are derived from the histiocytes. These tumors are relatively common in humans. Here we report a case of spontaneously-occurring malignant fibrous histiocytoma in an adult bonnet macaque.     

Nasal cavity carcinosarcoma in a bonnet macaque (Macaca radiata)

An example of a rare, highly aggressive malignant intranasal neoplasm in a male Bonnet macaque is presented. The tumor invaded the bony orbit, distorted the position of the globe, and metastasized to regional lymph nodes and lungs. Histologically the tumor was composed of both mesenchymal and epithelial components and probably was of minor salivary gland origin.     

Vocal communication of wild bonnet macaques (Macaca radiata)

Field observations and spectrographic analyses of sound recordings of South Indian bonnet macaques revealed a vocal repertoire of at least 25 basic patterns. The repertoire consists of well separated sound classes and acoustic categories connected by structural intergradation. Besides structural variations within and between different elements of the repertoire, the vocal system ofMacaca radiata is characterized by regular combinations of particular basic patterns. These combinations occurred not only between calls of similar structure and function but also between calls usually emitted in entirely different social contexts. According to the qualitative analysis, sex-specific asymmetries of the vocal behaviour were less pronounced than age-dependent characteristics. The comparison of clear call vocalizations ofMacaca radiata andM. fuscata revealed significant species-specific differences on the structural and the behavioural level. Evaluations of the structural features of alarm calls of various macaque species imply marked differences between members of thefascicularis group andsinica group on one hand and thesilenus group andarctoides group on the other.