Is There Any Association between Nutrition and Myopia? A Systematic Review.

SIGNIFICANCE: This systematic review highlights the possible role of nutrition in myopia based on qualitative analysis of vast and diverse literature that investigated this association. PURPOSE: We systematically reviewed the outcomes of the studies that previously investigated the association between nutrition and myopia. METHODS: EMBASE, MEDLINE, and PubMed were searched by two independent authors to identify cross-sectional, cohort, retrospective, or interventional studies that assessed the association of nutrition with myopia from inception to the year 2021. Furthermore, the reference list of the included articles was screened. The data from the included studies were extracted, and qualitative analysis was performed. Quality assessment for noninterventional studies and interventional trials was performed using the Newcastle-Ottawa Scale and Cochrane RoB 2, respectively. RESULTS: Twenty-seven articles were included in the review. Most of the nutrients and dietary elements investigated in noninterventional studies showed inconsistencies in their association with myopia, with the majority indicating no association. Nine studies showed a significant association of diverse nutrients and dietary elements with either an increase (odds ratio, 1.07) or a decrease (odds ratio, 0.5 to 0.96) in the risk of myopia development. However, a majority of these studies have minimal odds ratios with wider or overlapping confidence intervals, implicating weaker associations. All three nutrients and dietary elements assessed in the interventional trial had implications for myopia control, with two trials indicating a clinically minimal effect. CONCLUSIONS: This review implies that there is some evidence to indicate a potential influence of specific nutrients and dietary elements in myopia development, which are supported by several theories. However, given the vast, diverse, and complex nature of nutrition, more systematic investigation is warranted to comprehend the extent to which these specific nutrients and dietary elements are associated with myopia through longitudinal studies by subduing the limitations in the existing literature.

High stretchability, strength, and toughness of living cells enabled by hyperelastic vimentin intermediate filaments.

In many developmental and pathological processes, including cellular migration during normal development and invasion in cancer metastasis, cells are required to withstand severe deformations. The structural integrity of eukaryotic cells under small deformations has been known to depend on the cytoskeleton including actin filaments (F-actin), microtubules (MT), and intermediate filaments (IFs). However, it remains unclear how cells resist severe deformations since both F-actin and microtubules yield or disassemble under moderate strains. Using vimentin containing IFs (VIFs) as a model for studying the large family of IF proteins, we demonstrate that they dominate cytoplasmic mechanics and maintain cell viability at large deformations. Our results show that cytoskeletal VIFs form a stretchable, hyperelastic network in living cells. This network works synergistically with other cytoplasmic components, substantially enhancing the strength, stretchability, resilience, and toughness of cells. Moreover, we find the hyperelastic VIF network, together with other quickly recoverable cytoskeletal components, forms a mechanically robust structure which can mechanically recover after damage.

Synthesis, anti-HIV activity, integrase enzyme inhibition and molecular modeling of catechol, hydroquinone and quinol labdane analogs.

Labdane analogs with o-quinol, catechol and hydroquinone moiety have been synthesized using Diels-Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate, 3,4-dioxocyclohexa-1,5-diene-carboxylic acid and 3,6-dioxocyclohexa-1,4-dienecarboxylic acid with mono terpene 1,3-dienes, namely ocimene and myrcene. The resulting molecules and their derivatives were evaluated for their anti-HIV-1 activity using TZM-bl cell based virus infectivity assay. Two molecules 13 and 18 showed anti-HIV activity with IC50 values 5.0 (TI=11) and 4.6 (TI=46)µM, respectively. The compounds 17, 18 and 20 showed efficacy against HIV-1 integrase activity and showed inhibition with IC50 13.4, 11.1 and 11.5µM, respectively. The HIV-1 integrase inhibition activity of these synthetic molecules was comparable with integric acid, the natural fungal metabolite. Molecular modeling studies for the HIV-1 integrase inhibition of these active synthetic molecules indicated the binding to the active site residues of the enzyme.

Zona pellucida glycoproteins: Relevance in fertility and development of contraceptive vaccines.

Mammalian zona pellucida (ZP) is composed of three to four glycoproteins, which plays an important role during fertilization. Mutations in the genes encoding zona proteins are reported in women with empty follicle syndrome, degenerated oocytes and those with an abnormal or no ZP further emphasizing their relevance during fertility. Immunization with either native or recombinant ZP glycoproteins/proteins leads to curtailment of fertility in various animal species. Observed infertility is frequently associated with ovarian pathology characterized by follicular atresia and degenerative changes in ZP, which may be due to oophoritogenic T cell epitope(s) within ZP glycoproteins. To avoid ovarian dystrophy, B cell epitopes of ZP glycoproteins have been mapped by using bio-effective monoclonal antibodies. Immunization with the immunogens encompassing the mapped B cell epitopes by and large led to amelioration of follicular atresia. However, their use for human application will require more rigorous research to establish their safety and reversibility of the contraceptive effect. Nonetheless, to minimize human-animal conflicts, ZP-based contraceptive vaccines have been used successfully in the population management of free-ranging animal species such as feral horses, white-tailed deer and elephants. To control zoonotic diseases, attempts are also underway to control the population of other animal species including stray dogs, which acts as one of the major vectors for the rabies virus.

Myopia progression risk assessment score (MPRAS): a promising new tool for risk stratification.

Timely identification of individuals "at-risk" for myopia progression is the leading requisite for myopia practice as it aids in the decision of appropriate management. This study aimed to develop 'myopia progression risk assessment score' (MPRAS) based on multiple risk factors (10) to determine whether a myope is "at-risk" or "low-risk" for myopia progression. Two risk-score models (model-1: non-weightage, model-2: weightage) were developed. Ability of MPRAS to diagnose individual "at-risk" for myopia progression was compared against decision of five clinicians in 149 myopes, aged 6-29 years. Using model-1 (no-weightage), further 7 sub-models were created with varying number of risk factors in decreasing step-wise manner (1a: 10 factors to 1g: 4 factors). In random eye analysis for model-1, the highest Youden's J-index (0.63-0.65) led to the MPRAS cut-off score of 41.50-43.50 for 5 clinicians with a sensitivity ranging from 78 to 85% and specificity ranging from 79 to 87%. For this cut-off score, the mean area under the curve (AUC) between clinicians and the MPRAS model ranged from 0.89 to 0.90. Model-2 (weighted for few risk-factors) provided similar sensitivity, specificity, and AUC. Sub-model analysis revealed greater AUC with high sensitivity (89%) and specificity (94%) in model-1g that has 4 risk factors compared to other sub-models (1a-1f). All the MPRAS models showed good agreement with the clinician's decision in identifying individuals "at-risk" for myopia progression.

Mammalian zona pellucida glycoproteins: structure and function during fertilization.

Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 (Zp4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O-linked glycans of ZP3 in mice, N-linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis(x) sequence present on both N- and O-glycans of human ZP play an important role in human sperm-egg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the Gi protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.

Expanding and improving urban outreach immunization in Patna, India.

OBJECTIVES: We conducted a case study of an urban immunization outreach strategy to determine the feasibility of the intervention and to measure administrative immunization coverage outcomes. METHODS: A multipronged strategy for improving immunization coverage in Urban Patna, India, was implemented for 1 year (2009/2010). The strategy was designed to increase immunization sites, shift human resources, plan logistics, improve community mobilization, provide supervision, strengthen data flow and implement special vaccination drives. RESULTS: Over 1 year, the coverage of all primary vaccines of the Universal Immunization Program improved by over 100%. CONCLUSION: Coverage can be rapidly improved through outreach immunization in low socioeconomic areas if existing opportunities are carefully utilized.

Contraceptive efficacy of recombinant porcine zona proteins and fusion protein encompassing canine ZP3 fragment and GnRH in female beagle dogs.

PROBLEM: To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs. METHOD OF STUDY: Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs. RESULTS: Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control. CONCLUSION: These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.

Leukaemia inhibitory factor mediated proliferation of HTR-8/SVneo trophoblast cells is dependent on activation of extracellular signal-regulated kinase 1/2.

Leukaemia inhibitory factor (LIF) is one of the cytokines that is indispensable for embryo implantation. The aim of the present study was to investigate the role of activation of extracellular signal-regulated kinase (ERK) 1/2 in LIF-mediated proliferation of HTR-8/SVneo cells. Stimulation of HTR-8/SVneo cells with LIF (50 ng mL(-1)) resulted in an increase in cell proliferation (P < 0.05) via increased transition of cells to the G(2)/M phase of cell cycle. Stimulation with LIF resulted in the activation of both signal transducer and activator of transcription (STAT) 3 Tyr(705) and ERK1/2, but inhibition of ERK1/2 signalling by pretreatment of cells with U0126 (10 µM) for 2h resulted in abrogation of LIF-mediated increases in G(2)/M transition, with a significant decrease (P < 0.05) in absolute cell numbers compared with control. Although STAT3 silencing had no effect on LIF-dependent proliferation of HTR-8/SVneo cells, it did result in an increase in cell apoptosis, which increased further upon inhibition of ERK1/2 activation irrespective of LIF stimulation. Stimulation of cells with LIF increased the Bcl-2/Bax ratio, whereas ERK1/2 inhibition decreased the Bcl-2/Bax ratio, even after LIF stimulation. Hence, it can be inferred that ERK1/2 activation is essential for LIF-mediated increases in proliferation and that both STAT3 and ERK1/2 activation are important for the survival of HTR-8/SVneo cells.

Microbicides: a new hope for HIV prevention.

Human immunodeficiency virus (HIV), causative agent of acquired immunodeficiency syndrome (AIDS), is a global health concern. To control its transmission, safe sex has been proposed as one of the strategies. Microbicides- intravaginal/intrarectal topical formulations of anti-HIV agents have also been proposed to prevent HIV transmission. Microbicides would provide protection by directly inactivating HIV or preventing the attachment, entry or replication of HIV in susceptible target cells as well as their dissemination from target cells present in semen or the host cells lining the vaginal/rectal wall to other migratory cells. Microbicides must be safe, effective following vaginal or rectal administration, and should cause minimal or no genital symptoms or inflammations following long-term repeated usage. However, a safe and efficacious anti-HIV microbicide is not yet available despite the fact that more than 60 candidate agents have been identified to have in vitro activity against HIV, several of which have advanced to clinical testing. Nonetheless, proof-of-concept of microbicides has been established based on the results of recent CAPRISA 004 clinical trials. In this article, the trends and challenges in the development of effective and safe microbicides to combat HIV transmission are reviewed.

Regression of coronary atherosclerosis through healthy lifestyle in coronary artery disease patients--Mount Abu Open Heart Trial.

AIMS: To evaluate the efficacy of a unique healthy and happy lifestyle (HLS) program in regression of coronary atherosclerosis and reduction in cardiac events in an open trial. METHODS: One hundred and twenty three angiographically documented moderate to severe coronary artery disease (CAD) patients were administered HLS comprising of low-fat, high-fiber vegetarian diet, moderate aerobic exercise and stress-management through Rajyoga meditation. Its most salient feature was training in self-responsibility (heal+thy) and self-empowerment through inner-self consciousness (swasth; swa=innerself, sth=consciousness) approach using Rajyoga meditation. Following a seven day in-house sojourn, patients were invited for six month follow-up for reassessment and advanced training. At the end of two years, all patients were asked to undergo repeat angiography. RESULTS: Three hundred and sixty coronary lesions were analysed by two independent angiographers. In CAD patients with most adherence, percent diameter stenosis regressed by 18.23 +/- 12.04 absolute percentage points. 91% patients showed a trend towards regression and 51.4% lesions regressed by more than 10 absolute percentage points. The cardiac events in coronary artery disease patients were: 11 in most adherence, and 38 in least adherence over a follow-up period of 6.48 yrs. (risk ratio; most vs least adherence: 4.32; 95% CI: 1.69-11.705; P < 0.002). CONCLUSION: Overall healthy changes in cardiovascular, metabolic and psychological parameters, decline in absolute percent diameter coronary stenosis and cardiac events in patients of CAD were closely related to HLS adherence. However, more than 50% adherence is essential to achieve a significant change.

Safety of food crops on land contaminated with trace elements.

Contamination of agricultural soils with trace elements (TEs) through municipal and industrial wastes, atmospheric deposition and fertilisers is a matter of great global concern. Since TE accumulation in edible plant parts depends on soil characteristics, plant genotype and agricultural practices, those soil- and plant-specific options that restrict the entry of harmful TEs into the food chain to protect human and animal health are reviewed. Soil options such as in situ stabilisation of TEs in soils, changes in physicochemical parameters, fertiliser management, element interactions and agronomic practices reduce TE uptake by food crops. Furthermore, phytoremediation and solubilisation as alternative techniques to reduce TE concentrations in soils are also discussed. Among plant options, selection of species and cultivars, metabolic processes and microbial transformations in the rhizosphere can potentially affect TE uptake and distribution in plants. For this purpose, genetic variations are exploited to select cultivars with low uptake potential, especially low-cadmium accumulator wheat and rice cultivars. The microbial reduction of elements and transformations in the rhizosphere are other key players in the cycling of TEs that may offer the basis for a wide range of innovative biotechnological processes. It is thus concluded that appropriate combination of soil- and plant-specific options can minimise TE transfer to the food chain.

In humans, zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis.

BACKGROUND: It has been suggested that the zona pellucida (ZP) may mediate species-specific fertilization. In human the ZP is composed of four glycoproteins: ZP1, ZP2, ZP3 and ZP4. In the present study, the expression profile of ZP1 in human oocytes and ovaries, and its role during fertilization, is presented. METHODS: Human ZP1 (amino acid residues 26-551) was cloned and expressed in both non-glycosylated and glycosylated forms and its ability to bind to the capacitated human spermatozoa and to induce acrosomal exocytosis was studied. Monoclonal antibodies (MAbs), specific for human ZP1 and devoid of reactivity with ZP2, ZP3 and ZP4 were generated and used to localize native ZP1 in oocytes and ovarian tissues. RESULTS: The MAbs generated against ZP1 recognized specifically the zona matrix of secondary and antral follicles, ovulated oocytes, atretic follicles and degenerating intravascular oocytes, but failed to react with the Fallopian tube, endometrium, ectocervix and kidney. Escherichia coli and baculovirus-expressed recombinant human ZP1 revealed bands of approximately 75 and approximately 85 kDa, respectively, in western blot. Lectin binding studies revealed the presence of both N- and O-linked glycosylation in baculovirus-expressed ZP1. Fluorescein isothiocyanate-labelled E. coli- and baculovirus-expressed recombinant ZP1 bound to the anterior head of capacitated spermatozoa, however, only baculovirus-expressed ZP1 induced acrosomal exocytosis in capacitated sperm suggesting the importance of glycosylation in mediating the acrosome reaction. The human ZP1-mediated acrosome reaction involved the activation of both T- and L-type voltage-operated calcium channels, but does not activate the G(i)-coupled receptor pathway. Inhibition of protein kinase A and C significantly also reduced the ZP1-mediated induction of the acrosome reaction. CONCLUSION: These studies revealed for the first time that in humans ZP1, in addition to ZP3 and ZP4, binds to capacitated spermatozoa and induces acrosomal exocytosis.

Zona pellucida-induced acrosome reaction in human spermatozoa is potentiated by glycodelin-A via down-regulation of extracellular signal-regulated kinases and up-regulation of zona pellucida-induced calcium influx.

BACKGROUND: Glycodelin-A interacts with spermatozoa before fertilization, but its role in modulating sperm functions is not known. Zona pellucida-induced acrosome reaction is crucial to fertilization and its dysfunction is a cause of male infertility. We hypothesized that glycodelin-A, a glycoprotein found in the female reproductive tract, potentiates human spermatozoa for zona pellucida-induced acrosome reaction. METHODS: Glycodelin isoforms were immunoaffinity purified. The sperm intracellular cAMP concentration, protein kinase-A (PKA) and extracellular signal-regulated kinase (ERK) activities, and intracellular calcium were measured by ELISA, kinase activity assay kits and Fluo-4AM technique, respectively. The phosphorylation of inositol 1,4,5-trisphosphate type-1 receptor (IP3R1) mediated by ERK was determined by western blotting. Zona pellucida-induced acrosome reaction was detected by Pisum sativum staining. RESULTS: Pretreatment of spermatozoa with glycodelin-A significantly up-regulated adenylyl cyclase/PKA activity and down-regulated the activity of ERK and its phosphorylation of IP3R1, thereby enhancing zona pellucida-induced calcium influx and zona pellucida-induced acrosome reaction. Glycodelin-F or deglycosylated glycodelin-A did not have these actions. Treatment of spermatozoa with a protein kinase inhibitor abolished the priming activity of glycodelin-A, whilst ERK pathway inhibitors mimic the stimulatory effect of glycodelin-A on zona pellucida-induced acrosome reaction. CONCLUSIONS: Glycodelin-A in the female reproductive tract sensitizes spermatozoa for zona pellucida-induced acrosome reaction in a glycosylation-specific manner through activation of the adenylyl cyclase/PKA pathway, suppression of extracellular signal-regulated kinase activation and up-regulation of zona pellucida-induced calcium influx. The action of glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the zona pellucida.

'ZP domain' of human zona pellucida glycoprotein-1 binds to human spermatozoa and induces acrosomal exocytosis.

BACKGROUND: The human egg coat, zona pellucida (ZP), is composed of four glycoproteins designated as zona pellucida glycoprotein-1 (ZP1), -2 (ZP2), -3 (ZP3) and -4 (ZP4) respectively. The zona proteins possess the archetypal 'ZP domain', a signature domain comprised of approximately 260 amino acid (aa) residues. In the present manuscript, attempts have been made to delineate the functional significance of the 'ZP domain' module of human ZP1, corresponding to 273-551 aa fragment of human ZP1. METHODS: Baculovirus-expressed, nickel-nitrilotriacetic acid affinity chromatography purified 'ZP domain' of human ZP1 was employed to assess its capability to bind and subsequently induce acrosomal exocytosis in capacitated human spermatozoa using tetramethyl rhodamine isothiocyanate conjugated Pisum sativum Agglutinin in absence or presence of various pharmacological inhibitors. Binding characteristics of ZP1 'ZP domain' were assessed employing fluorescein isothiocyanate (FITC) labelled recombinant protein. RESULTS: SDS-PAGE and immunoblot characterization of the purified recombinant protein (both from cell lysate as well as culture supernatant) revealed a doublet ranging from ~35-40 kDa. FITC- labelled 'ZP domain' of ZP1 binds primarily to the acrosomal cap of the capacitated human spermatozoa. A dose dependent increase in acrosomal exocytosis was observed when capacitated sperm were incubated with recombinant 'ZP domain' of human ZP1. The acrosome reaction mediated by recombinant protein was independent of Gi protein-coupled receptor pathway, required extra cellular calcium and involved both T- and L-type voltage operated calcium channels. CONCLUSIONS: Results described in the present study suggest that the 'ZP domain' module of human ZP1 has functional activity and may have a role during fertilization in humans.

Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida.

BACKGROUND: Human egg is enveloped by a glycoproteinaceous matrix, zona pellucida (ZP), responsible for binding of the human spermatozoa to the egg and induction of acrosomal exocytosis in the spermatozoon bound to ZP. In the present manuscript, attempts have been made to delineate the downstream signalling components employed by human ZP to induce acrosome reaction. METHODS: Heat-solubilized human ZP (SIZP) was used to study the induction of acrosome reaction in capacitated human spermatozoa using tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA) in absence or presence of various pharmacological inhibitors. In addition, intracellular calcium ([Ca2+]i) levels in sperm using Fluo-3 acetoxymethyl ester as fluorescent probe were also estimated in response to SIZP. RESULTS: SIZP induces acrosomal exocytosis in capacitated human sperm in a dose dependent manner accompanied by an increase in [Ca2+]i. Human SIZP mediated induction of acrosome reaction depends on extracellular Ca2+ and involves activation of Gi protein-coupled receptor, tyrosine kinase, protein kinases A & C and phosphoinositide 3 (PI3)- kinase. In addition, T-type voltage operated calcium channels and GABA-A receptor associated chloride (Cl-) channels play an important role in SIZP mediated induction of acrosome reaction. CONCLUSIONS: Results described in the present study provide a comprehensive account of the various downstream signalling components associated with human ZP mediated acrosome reaction.

Vaccines for immunological control of fertility.

Vaccines have been proposed as one of the strategies for population control. Immunocontraceptive vaccines can be designed to inhibit: (1) production of gametes (sperm and egg); (2) functions of gametes, leading to blocking of fertilization; and (3) gamete outcome (pregnancy). Immunization with gonadotropin-releasing hormone coupled to different carriers has shown curtailment in the production of sperm with concomitant infertility in various species. Immunization of nonhuman primates and men with ovine follicle stimulating hormone has also resulted in reduced sperm output. Various spermatozoa-specific proteins such as FA1, PH-20, LDH-C4, SP-10, SP-17, sp56, SPAG9, and Izumo have been proposed as candidate antigens to develop contraceptive vaccines, which have shown efficacy in inhibiting fertility in different animal models. Immunization with zona pellucida glycoproteins-based immunogens also results in curtailment of fertility in a variety of species. However, ways to overcome the observed oophoritis associated with zona proteins immunization have yet to be discovered, a necessary step before their proposal for control of human population. Nonetheless, this is a very promising approach to control wildlife animal population. Phase II clinical trials of ß-human chorionic gonadotropin-based vaccine in women have established the proof of principle that it is possible to inhibit fertility without any untoward side-effects by vaccination. Further scientific inputs are required to increase the efficacy of contraceptive vaccines and establish their safety beyond doubt, before they can become applicable for control of fertility in humans.

Immune physiology and oogenesis in fetal and adult humans, ovarian infertility, and totipotency of adult ovarian stem cells.

It is still widely believed that while oocytes in invertebrates and lower vertebrates are periodically renewed throughout life, oocytes in humans and higher vertebrates are formed only during the fetal/perinatal period. However, this dogma is questioned, and clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal from ovarian stem cells (OSCs) in adult human ovaries, and of the role of third-party bone marrow-derived cells (monocyte-derived tissue macrophages and T lymphocytes) could help provide a better understanding of the causes of ovarian infertility, its prevention, and potential treatment. We have reported differentiation of distinct cell types from OSC and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. OSCs are also capable of producing neural/neuronal cells in vitro after sequential stimulation with sex steroid combinations. Hence, OSC represent a unique type of totipotent adult stem cells, which could be utilized for autologous treatment of premature ovarian failure and also for autologous stem cell therapy of neurodegenerative diseases without use of allogeneic embryonic stem cells or somatic cell nuclear transfer. The in vivo application of sex steroid combinations may augment the proliferation of existing neural stem cells and their differentiation into mature neuronal cells (systemic regenerative therapy). Such treatment may also stimulate the transdifferentiation of autologous neural stem cell precursors into neural stem cells useful for topical or systemic regenerative treatment.

Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian follicles.

The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology.

Study origin of germ cells and formation of new primary follicles in adult human and rat ovaries.

The central thesis regarding the human ovaries is that, although primordial germ cells in embryonal ovaries are of extraovarian origin, those generated during the fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE) bipotent cells. With the assistance of immune system-related cells, secondary germ cells and primitive granulosa cells originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly to be recognized as self-structures and renewed later. With the onset of menarche, a periodical oocyte and follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs for healthy babies are always available during the prime reproductive period. The periodical follicular renewal ceases between 35 and 40 yr of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. The human OSE stem cells preserve the character of embryonic stem cells, and they may produce distinct cell types, including new eggs in vitro, particularly when derived from patients with premature ovarian failure or aging and postmenopausal ovaries. Our observations also indicate that there are substantial differences in follicular renewal between adult human and rat ovaries. As part of this chapter, we present in detail protocols utilized to analyze oogenesis in humans and to study interspecies differences when compared to the ovaries of rat females.