Molecular approaches to contraceptive development.

The next generation of contraceptives will be based on the identification of novel molecules essential for reproductive processes and will rely on the refinement of older as well as newer technologies. Functional analysis of naturally occurring reproductive genetic disorders and creation of mice null for specific genes would greatly assist in the choice of genetic targets for contraceptive development. Structure-based design of drugs as exemplified by the preparation of an orally active non-peptide gonadotropin releasing hormone (GnRH) would revolutionize drug formulation and delivery for a peptide analogue. This review examines some of the molecular targets that may change contraceptive choices in the future.

Partial cloning and sequencing of a cDNA encoding bonnet monkey (Macaca radiata) oviduct specific protein.

Based on the complete nucleic acid sequence of human estrogen dependent oviductal protein and deduced amino acid sequence, potential antigenic site of the protein was identified. Two oligonucleotide primers were designed to specifically amplify the region which includes this antigenic site. With the expectation that the human, and monkey oviductins would have high nucleotide sequence homology, Bonnet monkey oviduct along with endometrium was obtained on day 5, 9, 12 and 22 of the cycle. Using RT PCR correct sized PCR product was detected in oviduct taken from day 9 and 12 of the cycle. PCR product was cloned into pBluescript KS[+] and nucleic acid sequence determined. A 96% homology to human, baboon and rhesus monkey estrogen induced glycoprotein, and a 84-88% homology to other mammalian oviductal protein was noted, thus confirming the authenticity of cDNA clone for monkey fallopian tube specific protein.

Characterization and localization of estrogen and progesterone receptors of human fallopian tube.

The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopian tube was investigated by immunohistochemical localization with specific monoclonal antibodies. Nuclear immunostaining was observed. Intense PR immunostaining was seen in tissues obtained at mid cycle and luteal stages of the normal menstrual cycle. On the other hand, enhanced staining for ER was seen in early follicular phase and mid cycle. Menopausal tissues showed negligible staining for both ER and PR. The ER and PR were characterized for their molecular size, anatomical distribution and levels during the menstrual cycle and in menopause. ER protein was present throughout the cycle and also during menopause. Western blot analysis revealed two forms of ER approximately 66 kDa and a truncated from approximately 49 kDa in hFT. Presence of A [approximately 90 kDa] and B [approximately 120 kDa] isoforms of human PR was detected. Follicular and early luteal tissue possessed relatively high concentration of immunoreactive PR whereas it was almost undetectable in menopausal tissues. These results suggests that ER and PR are regulated by the changing ovarian steroid hormones.

Effect of estrogen and progesterone on newly synthesised and released human fallopian tube specific proteins.

Current study was carried out to identify the profile of newly synthesized and released proteins by human fallopian tube (hFT). Results indicated that hFT during menopause synthesised and released only 2-3 proteins as against several proteins ranging from molecular weight (MW) approximately 20 to approximately 130 kD during normal menstrual cycle. In vitro addition of estradiol-17 beta (E2) resulted in synthesis and release of a number of proteins including specific protein of MW 110-130 kD. Addition of progesterone (P) however, led to inhibition of protein synthesis and a combination of E2 and P negated the effect of the latter. An alteration in oviductal secretory protein-profile following addition of E2 in vitro were similar to that observed during normal menstrual cycle.

Immunoreactive riboflavin carrier protein concentration during ovulatory cycle in the common marmoset (Callithrix jachhus): role of estrogen.

Common marmoset, a non-human primate, is increasingly being used as a model system in reproductive endocrinology. Marmosets do not menstruate but have normal cycle length of 28 +/- 2 days. The presence of protein immunologically and functionally similar to the well characterized chicken riboflavin carrier protein (cRCP) has been demonstrated in circulation in pregnant marmosets. Marmoset RCP (mRCP) has been partially purified and the molecular weight of the immunoreactive protein is similar to cRCP. The source of RCP in rodents appears to be maternal liver, suggesting thereby that RCP levels could be modulated by changing hormonal pattern that occurs during the cycle. To study the hormonal control of marmoset RCP, immunoreactive RCP levels were monitored during normal cycle. Plasma progesterone was estimated by RIA and mRCP was measured using a heterologous cRCP RIA. When mRCP and progesterone were analysed for a period of 40 days (n = 5) in adult females, very low amount of mRCP was measured during the luteal phase and a single sharp peak was observed during the follicular phase of the cycle. These data suggested that the induction of mRCP was regulated by estrogen. Direct evidence for the role of estrogen in the elaboration of RCP, was the observation that exogenous administration of estradiol 17 B to immature marmosets resulted in a time and dose dependent increase in plasma RCP levels. As early as 24 hr following hormonal administration, a measurable increase in mRCP with a maximum increase, 5-fold over the zero period was seen 3 days following a single administration of estradiol 17 B (5 mg/kg body wt). RCP levels declined thereafter and was indistinguishable by day 7. These studies indicate that the marmoset RCP levels are regulated by estrogen.

Improved presentation of antigenic sites in enzyme-linked immunosorbent assay: antigenicity of reduced and carboxymethylated riboflavin carrier protein.

Disulphide reduced and carboxymethylated riboflavin carrier protein (RCM-RCP), an unfolded derivative of chicken RCP, does not bind riboflavin and there is a drastic reduction in its ability to interact with antiserum to cRCP. Antibodies to RCM-RCP are directed against sequential epitopes(s). On radioiodination of RCM-RCP, a maximum of 30-50% binding at dilution of 1:500 was obtained with rabbit antibodies RCM-RCP [n = 5]. However, high titer antibodies was obtained when radioiodinated RCM-RCP was immobilized on ELISA microtiter plates suggesting that immobilization of RCM-RCP leads to either preservation of antigenic sites or improved presentation of the antigenic determinants. An avidin-biotin system was utilized to develop an ELISA.

Termination of pregnancy in mice following antiserum administration to modified chicken riboflavin carrier protein.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards conformational epitopes and antibodies to disulphide bond reduced carboxymethylated RCP (RCM-RCP) are towards sequential epitopes. The major cyanogen bromide (CNBr) fragments and tryptic fragments of RCM-RCP interact with both antiserum to RCM-RCP and RCP. Passive immunization of pregnant mice with antibodies to RCM-RCP results in bioneutralization, leading to termination of pregnancy. Recently, a major tryptic fragment of RCM-RCP (24 +/- 2 kd) which could assume conformation at the antibody combining site of native RCP, obtained following mild trypsinization has been identified [Natraj et al. J. Biosci, 15 (1990) 341]. Rabbit antibodies to RCM-RCP treated with trypsin generated antibodies of low titer which interacted with RCM-RCP as well as RCP. The interaction of this antibody with RCP was of high affinity and could be displaced with RCP. The bioneutralizing ability of the antibody was demonstrated by its ability to cause termination of pregnancy in mice.

Enzyme-linked immunosorbent assay for riboflavin carrier protein and modified protein: application for epitope analysis.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards the conformational epitopes and antibodies to disulphide bond reduced carboxymethylated riboflavin carrier protein (RCM-RCP) to the sequential epitopes. Taking advantage of this premise and in order to map the epitopes of RCP recognized by the antibodies, enzyme-linked immunosorbent assays were validated for RCP and RCM-RCP using the Avidin-Biotin system. The usefulness of these assays were illustrated when antigenicity of peptides derived from RCM-RCP following trypsinization were examined. Two major (T1,T2) and one minor peptide (T3) fractions were obtained when the tryptic peptides were fractionated on DEAE-cellulose. RCP has a blocked N-terminal. Tryptic peptides (T1 and T2) on microsequencing revealed the absence of an N-terminal amino acid, indicating that these fragments emanate from the N-terminal region of RCP. In support of this observation is the finding that antipeptide antibody to cRCP (10-24) of cRCP interacted with T1 as well as T2 indicating the presence of the sequential epitope (10-24) of cRCP in these fragments. In RCP-ELISA, only T2 displaced RCP and peptides T1 and T2 displaced RCM-RCP in RCM-RCP ELISA. Differences in the ability of these fragments (T1 and T2) to displace RCP and RCM-RCP reflect the subtle changes in the spatial structures of these epitopes in RCP and RCM-RCP.