Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization.

Association of lactate, intracellular pH, and intracellular calcium during capacitation and acrosome reaction: contribution of hamster sperm dihydrolipoamide dehydrogenase, the E3 subunit of pyruvate dehydrogenase complex.

The role of dihydrolipoamide dehydrogenase (DLD), the E3 subunit of the pyruvate dehydrogenase complex (PDHc) in hamster sperm capacitation and acrosome reaction has been implicated previously. In this study, attempt has been made to understand DLD/PDHc involvement from the perspective of pyruvate/lactate metabolism. Inhibition of DLD was achieved by the use of a specific inhibitor, 5-methoxyindole-2-carboxylic acid. It was seen that 5-methoxyindole-2-carboxylic acid-treated spermatozoa with inhibited DLD (and PDHc) activity had lactate accumulation, which caused an initial lowering of the intracellular pH and calcium and an eventual block in capacitation and acrosome reaction. Collectively, the data reveal a significant contribution of the metabolic enzymes DLD and PDHc to lactate regulation in hamster spermatozoa during capacitation and acrosome reaction. Additionally, the importance of lactate regulation in the maintenance of sperm intracellular pH and calcium, two important physiologic factors essential for sperm capacitation and acrosome reaction, has also been established.

Perspectives of contraceptive choices for men.

Apart from condoms and vasectomy, which have several limitations of their own, no other methods of contraception are available to men. Various chemical, hormonal, vas based and herbal contraceptives have been examined and few of them have reached the stage of clinical testing. Promising leads have been obtained from testosterone buciclate/undecanoate, alone or in combination with levonorgestrel butanoate or cyproterone acetate, RISUG, an injectable intravasal contraceptive and a few herbal products, particularly the seed products of Carica papaya. It is feasible that an ideal male contraceptive, that meets out all the essential criteria will be made available to the community in the near future.

Safety evaluation of long-term vas occlusion with styrene maleic anhydride and its non-invasive reversal on accessory reproductive organs in langurs.

AIM: To evaluate the safety of the long term vas occlusion with styrene maleic anhydride (SMA) and its non-invasive reversal at the level of accessory reproductive glands ARGs in langurs. METHODS: The morphology of seminal vesicle and ventral prostate was evaluated by light as well as transmission electron microscopy. Serum clinical chemistry and urine albumin were evaluated in an autoanalyzer using reagent kits. Fructose, acid phosphatase and zinc in the seminal plasma were evaluated spectrophotometrically according to the WHO manual. Serum testosterone, prostate specific antigen and sperm antibodies were evaluated by enzyme-linked immunosorbent assays (ELISA) using reagent kits and hematology was estimated according to standard procedures. RESULTS: The morphological features and secretory activity of the seminal vesicle and prostate were normal as evidenced by the presence of well-developed mitochondria, rough endoplasmic reticulum, Golgi bodies, secretory granules and normal nuclear characteristics throughout the course of investigation. Serum testosterone and prostate specific antigen remained unaltered and serum antisperm antibodies level presented negative titres. Urine albumin was nil. Total red blood corpuscles (RBC), white blood corpuscles (WBC), hemoglobin (Hb) and red cell indices, serum protein, glucose, cholesterol, creatinine, creatine kinase (CK), serum glutamate oxalate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), lactate dehydrogenase (LDH), bilirubin, urea, triglycerides and high-density lipoprotein (HDL) did not show appreciable changes following vas occlusion and after its non-invasive reversal. Although fructose, acid phosphatase (ACP) and zinc in the seminal plasma showed a significant reduction following vas occlusion, it could not be related to the morphology of seminal vesicle and prostate. CONCLUSION: SMA vas occlusion and its non-invasive reversal do not damage the accessory reproductive organs.

Efficacy trial on the purified compounds of the seeds of Carica papaya for male contraception in albino rat.

The contraceptive efficacy and toxicological screening of the two principal compounds, MCP I and ECP I, isolated from the seeds of Carica papaya, in male albino rats at the standardized dose regimen, at 50 mg/kg b.w./day, for a period of 360 days and up to 90 days of treatment withdrawal have been reported. The body and organ weights, cauda epididymal sperm characteristics, androgen sensitive tissue biochemistry, reactive oxygen species and anti-oxidant defense system in the cauda epididymal microenvironment, histology and ultrastructure of testis and cauda epididymis, histology of seminal vesicle and prostate, toxicological investigations through routine hematology and serum clinical chemistry, sexual behaviour and fertility index have been studied. The results revealed that oral administration of MCP I and ECP I were equally effective, exhibiting complete inhibition of sperm motility following 90 days of treatment that coincided with a gradual and significant decline in cauda epididymal sperm density, percent viable spermatozoa and significant increase in sperm anomalies. Histology of testis of treated animals revealed degenerated germinal epithelium, vacuolization in Sertoli cells and proliferating germ cells and disturbances in spermatid differentiation. Spermatogonial stem cell reserves and Leydig cells appeared normal. Ultrastructure of the testis revealed vacuolization in the Sertoli cells and germ cells, loss of cytoplasmic characteristics in the Sertoli cells, nuclear degeneration and mitochondrial vacuolization in spermatocytes and spermatids. Leydig cells exhibited steroidogenic features. Cauda epididymis showed normal epithelial cell function. Absence of spermatozoa or disruption of spermatozoa clusters in the lumen were evident. Ultrastructure of cauda epididymis revealed normal secretory activity. Morphology of seminal vesicle and prostate of the treated animals were comparable to control animals. Serum testosterone, tissue biochemical and toxicological parameters remained unaffected. Fertility test revealed 100% efficacy. All the altered parameters showed sign of recovery following 90 days of treatment withdrawal. It is concluded that both MCP I and ECP I are equally effective in terms of contraceptive efficacy which is likely reversible and without adverse side effects.

Ultrastructural changes in the testis and epididymis of rats following treatment with the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya.

The benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya at a dose of 10 mg/rat/day for 150 days, which has shown a total inhibition of motility, reduced sperm count and infertility, was tested to de fi ne the mode of action at the subcellular level in the testis and epididymis. The ultrastructure of the testis of the treated animals revealed no appreciable changes in the subcellular characteristics. The mechanism of protein synthesis as well as steroidogenesis were evident in the Sertoli cells while the spermatogonia, spermatocytes and spermatids, both round and elongated, depicted a prominent nucleus, distinct nuclear membrane and cytoplasmic characteristics indicating normal germ cell differentiation. The principal cells of the cauda epididymis were characterized by the presence of well-de fi ned rough endoplasmic reticulum, mitochondria, Golgi bodies and secretary granules, suggesting active secretory functions. The absorptive function of the cauda epididymis was evidenced by the presence of numerous vesicles and multivesicular bodies adjacent to stereocilia. It is concluded that the inhibition of sperm motility by the drug could be due to other epididymal factors rather than the subcellular characteristics of testis and epididymis.

Status of spermatogenesis and sperm parameters in langur monkeys following long-term vas occlusion with styrene maleic anhydride.

Vas occlusion by styrene maleic anhydride (SMA), trade name RISUG (one of the promising male contraceptive procedures currently in phase III clinical trials), at 60 mg/vas deferens dissolved in 120 micro L dimethyl sulphoxide (DMSO) at up to a 540-day study period caused severe oligospermia in the first 2 to 3 ejaculations and uniform azoospermia in the subsequent ejaculations without toxicity in langur monkeys. The ejaculated spermatozoa were necroasthenoteratozoospermic, suggesting instant sterility. Routine hematology and clinical chemistry parameters and the serum testosterone and sperm antibody titers remained unchanged from their pretreatment values until 540 days vas occlusion. Histology of testes revealed continued spermatogenesis throughout the study period. The stages of spermatogenesis appeared normal until 300 days of vas occlusion. At 360 days of vas occlusion, germ cells appeared in the lumen. Degeneration of seminiferous epithelium was evident in some of the tubules. Following 420 days of vas occlusion, the central portion of the testis showed regressed seminiferous tubules depicting various shapes and devoid of germ cells, which continued until 540 days of vas occlusion. Ultrastructure of the testes after 540 days of vas occlusion revealed vacuolization in the cytoplasm of Sertoli cells and degenerative features in the membranes of the spermatocytes and spermatids in the affected seminiferous tubules. The sub-cellular features of the normal tubules were similar to those of controls. The results suggest focal degeneration of seminiferous epithelium in the central portion of the testis following long-term vas occlusion with SMA.

Chloroform extract of Carica papaya seeds induces long-term reversible azoospermia in langur monkey.

AIM: To evaluate the antifertility activity of the chloroform extract of Carica papaya seeds by oral administration in langur monkey, Presbytis entellus entellus. METHODS: The chloroform extract of Carica papaya seeds, 50 mg/kg/day, was administered orally for 360 days to adult male langur monkeys. The sperm characteristics by light and electron microscopy, the sperm functional tests, the semen biochemistry, the serum testosterone level, the Leydig cell function, and the histology and ultrastructure of testis were determined to evaluate the antifertility activity and the blood biochemistry and hematology, to evaluate the toxicology. RESULTS: The extract gradually decreased the sperm concentration since days 30-60 of treatment with a total inhibition of sperm motility, a decrease in sperm viability and increase in sperm abnormality. Azoospermia was observed after day 90 of treatment and continued during the whole treatment period. Treatment withdrawal resulted in a gradual recovery in these parameters and 150 days later they reverted to nearly the pretreatment values. Morphological observation of the ejaculated sperm by light and scanning electron microscopy showed deleterious changes, particularly on the mid-piece. Sperm functional tests, viz., sperm mitochondrial activity index, acrosome intactness test and hypo-osmotic swelling test scored in the infertile range during treatment and returned to the fertile values 150 days after drug withdrawal. Histology of the testis revealed shrunken tubules, germ cell atrophy and normal Leydig cells. Ultrastructure of the testis showed vacuolization in the cytoplasm of Sertoli cells and germ cells. Loss of cytoplasmic organelles were evident in the spermatocytes and spermatids. Round spermatids showed loss of Golgi bodies, peripheral mitochondria and vacuolated cytoplasm, indicating maturational arrest. Leydig cell functional test indicated a mild inhibition of steroidogenic function. Haematology and serum biochemistry study disclosed no significant toxicological effect and the serum testosterone level was not affected. CONCLUSION: Carica papaya seed extract may selectively act on the developing germ cells, possibly mediated via Sertoli cells, leading to azoospermia.