Seasonal changes in the hypothalamo-hypophyseal-ovarian system in the catfish,Heteropneustes fossilis (Bloch)*

The annual reproductive cycle of the catfish, H. fossilis (Bloch) is divided into the preparatory period (February-April), the prespawning period (May-June), the spawning period (July-August) and the postspawning period (September-January). During the early postspawning period (September-November), the hypothalamo-hypophyseal-ovarian system shows a gradual regression. In January, the hypothalamic nuclei, the pars magnocellularis (PMC), the pars parvocellularis (PPC) of the nucleus preopticus (NPO), and the nucleus lateralis tuberis (NLT) show renewed activity, as shown by a significant increase in their nuclear diameters and an accumulation of neurosecretory material (NSM) in their cell bodies. The hypophysis and the ovary remain quiescent. During the preparatory period, all the hypothalamic neurons studied indicate decreased activity but simultaneously show an accumulation of NSM in their cell bodies. The number of granulated basophils in the proximal pars distalis (PPD) of the hypophysis remains low but ovarian weights increase, presumably due to the multiplication of oogonia. In the prespawning period, there is a marked accumulation of NSM in the cell bodies of the hypothalamic neurons and at the same time the number of granulated basophils in the PPD of the hypophysis dramatically increases with concomitant increase in vitellogenic activity in the ovary. During the spawning period, the hypothalamic neurons continue to store NSM in their cell bodies and simultaneously there is a tremendous increase in the number of granulated basophils in the PPD of the hypophysis and the ovary has a large proportion of yolky primary oocytes. Spawning is associated with a significant degranulation of the granulated basophils in the PPD of the hypophysis. The significance of the results is discussed in relation to the environmental and hormonal regulation of seasonal ovarian activity.     

Effect of long photoperiod on the maturation of ovary of the catfish, Mystus tengara (Ham.)

A study has been made of the effect of photoperiodism on the ovarian maturation of fresh water teleost, Mystus tengara (Ham.). The magnitude of ovarian recrudescence is more during maturing virgin phase as compared to during immature virgin and preparatory virgin phases in long photoperiod (14 hours/day) treated fish. Thus, the response of the ovary to long photoperiod increases with the approach of natural spawning period. The mature eggs are more in long photoperiod treated fish than in controls exposed to 12 hours/day photoperiod. In long photoperiod treated fish, prespawning period is reduced considerably. During spawning and post-spawning periods, the ovaries are refractory, since long photoperiod is ineffective in maintaining gravid ovaries beyond spawning period and the ovarian regression during post spawning period sets in regardless of nature of photoperiod treatment.     

Seasonal fluctuation in lipid and cholesterol content of ovary, liver and blood serum in relation to annual sexual cycle in Heteropneustes fossilis (Bloch).

Seasonal fluctuation in lipid content of ovary and liver and cholesterol content of ovary, liver and serum in relation to annual reproductive cycle in H. fossilis has been worked out. Reduction in lipid content of liver was associated with decrease in hepatosomatic index (HSI) in prespawning and spawning phases. But there was an obvious increase in the lipid level of ovary in the above phases which coincided with enhanced gonadosomatic index (GSI) records. Serum and ovary showed a subsidence in their cholesterol contents in prespawning followed by increased level during spawning period. Substraction in liver cholesterol started in prespawning and continued till spawning phase and thereafter regained upward trend. Lipid and cholesterol levels in all organs studied along with GSI and HSI returned to initial values by the end of postspawning stage. Administration of clomid, sexovid, PGE1 and PGF2 alpha for a week did not induce any change in HSI, GSI, lipid and cholesterol values of liver and serum. However, these drugs were effective in suppressing the ovarian cholesterol within a week. The above drugs seem to affect cholesterol level indirectly since they trigger gonadotrophin secretion which in turn probably accelerated cholesterol utilization in ovary for increased steroid hormone output.     

Some effects of external factors upon the reproductive behavior of the grass frog Rana t. temporaria L. (Ranidae,Anura)

Summary The reproductive period of Rana temporaria can be subdivided into a prespawning period and a spawning period. During the prespawning period only a few Rana temporaria, most of them males, are active in the spawning biotope. There is no calling activity, and deposition of spawn rarely occurs. The time of onset of the prespawning period is variable. In this period the frogs are highly responsive to a drop in the temperature of air and water, burying themselves in the bottom or leaving the spawning waters.By contrast, many animals participate in the spawning period. Migration to the spawning ground takes place chiefly at night. The frogs engage in mating activity immediately upon arrival, and both males and females depart as soon as they have spawned, migrating away in directions corresponding to those of the immigration. During the spawning period there is no demonstrable response of the grass frogs to weather conditions. Rana temporaria begins its spawning period at the so-called set time only in meteorologically normal years. When meteorological conditions are unusual, the spawning period can begin as much as 10 days earlier or later.Possible external timing signals for the onset of migration include temperature in combination with day length, and a drop in atmospheric pressure accompanied by rainfall.Herrn Professor Dr. Rolf Danneel zur Vollendung des 75. Lebensjahres gewidmet     

Seasonal effects of melatonin on ovary and plasma gonadotropin and vitellogenin levels in intact and pinealectomized catfish, Clarias batrachus (Linn)

Effects of daily administration of melatonin for 15 days were evaluated with respect to ovarian activities and plasma gonadotropin (GtH II) and vitellogenin (Vg) levels in intact (INT) and pinealectomized (Px) female catfish, C. batrachus, during preparatory (April), prespawning (May and June), spawning (July) and post-spawning (September) periods. Px (saline control groups) caused a stimulatory effect during preparatory (with respect to Vg synthesis and incorporation) and prespawning (with respect to Vg synthesis) periods whereas no effect was observed during spawning and post-spawning periods with respect to the reproductive parameters studied. During April, melatonin-treatment significantly decreased plasma GtH II levels and percentage of vitellogenic oocytes without any significant changes in plasma Vg levels and gonadosomatic index (GSI). During early prespawning period, in May, 50microg melatonin brought about a significant reduction in plasma GtH II levels in INT group, whereas 100microg caused a decrease in all parameters; on the other hand, in Px groups both dose levels proved to be inhibitory. In June (late prespawning period) melatonin-treatment could not bring about any change in GSI and plasma Vg levels compared to the control groups regardless of Px but plasma GtH II and mean number of yolky oocytes were significantly reduced in melatonin-treated INT group. During spawning period (July) melatonin inhibited the GSI, mean number of yolky oocytes and plasma GtH II levels without affecting plasma Vg levels. In September (post-spawning period), melatonin did inhibit both GSI and plasma GtH II levels. The results, thus, indicate that melatonin showed variable effects (inhibitory and/or no effect) to GSI, mean number of yolky oocytes and plasma Vg levels but a consistent inhibiton of plasma GtH II levels indicating that melatonin may control the reproduction by blocking the GtH II release from the pituitary via affecting the hypothalamo-hypophysial axis.     

Stage-specific initiation of milk synthesis in the ovariectomized pregnant mouse

Stage-specific initiation of milk synthesis was investigated in mice ovariectomized on days 7, 9, 11 and 13 of pregnancy, and the lactogenic response of the mammary gland was monitored by using the synthesis of casein, lactose content and PRL binding as parameters. Pregnant mice ovariectomized on day 7 or 9 showed no increase in these parameters 24 h after operation. After day 11 of pregnancy, a clear response was induced by ovariectomy, characterized by a low level of casein synthesis, small amounts of lactose and PRL binding, which increased linearly in a time-dependent manner thereafter. These results indicate that pregnant mice after 11 days are able to initiate synthesis of casein and lactose in parallel. Mammary glands were cultured with insulin, cortisol and PRL. The mammary explants of pregnant mice at 7 and 9 days showed active synthesis of casein after a lag period of 2 days, suggesting that the mammary gland in early pregnancy is less sensitive to PRL, probably due to the absence of an increase in PRL binding after ovariectomy.     

Fecundity regulation by atresia in turbot Scophthalmus maximus in the Baltic Sea

Down‐regulation of fecundity through oocyte resorption was assessed in Baltic Sea turbot Scophthalmus maximus at three locations in the period from late vitellogenesis in April to spawning during June to July. The mean ± s.d . total length of the sampled fish was 32·7 ± 3·1 cm and mean ± s.d . age was 6·2 ± 1·5 years. Measurements of atresia were performed using the ‘profile method’ with the intensity of atresia adjusted according to the ‘dissector method’ (10·6% adjustment; coefficient of determination was 0·675 between methods). Both prevalence (portion of fish with atresia) and intensity (calculated as the average proportion of atretic cells in fish displaying atresia) of atresia were low in prespawning fish, but high from onset of spawning throughout the spawning period. Atretic oocytes categorized as in early alpha and in late alpha state occurred irrespective of maturity stage from late prespawning individuals up to late spawning fish, showing that oocytes may become atretic throughout the spawning period. Observed prevalence of atresia throughout the spawning period was almost 40% with an intensity of c. 20%. This indicates extensive down‐regulation, i.e. considerably lower realized (number of eggs spawned) v. potential fecundity (number of developing oocytes), suggesting significant variability in reproductive potential. The extent of fecundity regulation in relation to fish condition (Fulton's condition factor) is discussed, suggesting an association between levels of atresia and fish condition.     

Progesterone administration prolongs the inhibitory effects of hyperprolactinemia on luteinizing hormone secretion in acutely ovariectomized rats

Several studies have shown that hyperprolactinemia in rats inhibits the post-gonadectomy rise in plasma luteinizing hormone (LH) for a limited period only. In intact rats the suppression of plasma LH during hyperprolactinemia is more prolonged. In the present study we have examined the possibility that the elevated levels of progesterone brought about by the raised plasma prolactin levels in intact rats are involved in the maintenance of LH inhibition. We have observed the effect of exogenous progesterone administration during the early post-ovariectomy period on plasma LH levels in female rats made hyperprolactinemic by administration of the dopamine antagonist, domperidone. Following ovariectomy of virgin, female rats, plasma LH was determined on each day from Day 3 to Day 10 after ovariectomy. In control rats plasma LH had increased by approximately 5-fold during the period of the experiment. In control rats treated with progesterone the rise in plasma LH was inhibited temporarily but LH had increased to similar levels to the controls by Day 10. In hyperprolactinemic rats LH was suppressed until Day 7, after which significant rises were observed. However, in hyperprolactinemic rats treated with progesterone, LH did not rise in a similar fashion, and remained low throughout the experiment. We conclude that a combination of hyperprolactinemia and raised plasma progesterone concentrations is necessary for the continued inhibition of LH release after ovariectomy.     

Sex-specific metabolic changes in the annual reproductive cycle of a freshwater catfish

Sex-specific enzymatic and other biochemical changes were studied in the annual reproductive cycle of the freshwater catfish, Clarias batrachus. Citrate synthase (CS) activity of brain, liver and skeletal muscle was maximum in spawning and minimum in postspawning showing a sharp decline in aerobic capacity after spawning. Similar CS activity in remaining phases of the annual reproductive cycle reflects similar energy need during regressed, preparatory and prespawning phases. Glucose 6-phosphate dehydrogenase (G6-PDH) activity declined in spawning and postspawning indicating a possible decrease in lipid and nucleic acid syntheses. The subsequent increase in G6-PDH activity with onset of resting and maintenance of the increased level throughout preparatory and prespawning phases shows restoration of biosynthetic activity. Higher activity of G6-PDH in female than male may be to satisfy a greater biosynthetic need of female reproduction and breeding. The decreased RNA content of tissues showed reduction in protein synthesis capacity during spawning and subsequent increase through postspawning until resting phase. The RNA content of brain and liver was higher in female than male during preparatory and prespawning, which may be associated with higher protein synthesis requirement of female for preparation of reproductive activities. The requirement based sex related changes in metabolism of catfish may be enzyme, tissue or reproductive phase-specific.     

Castration-induced hyperactivity of seminal vesicle in the catfish Clarias batrachus: a case of paradox and blockade by antiandrogen (cyproterone acetate) treatment

Castration of the catfish Clarias batrachus in late preparatory-early prespawning phase (April-May) caused time-dependent stimulatory effect on morphology, weight, and in the concentrations of biochemical correlates, such as total proteins, fructose, hexosamines and sialic acid in the seminal vesicle (SV). The peak changes were noticed on week 4 of castration. The hyperactivity was related to augmented production of testosterone by the SV of castrates with the levels significantly high from week 3 onwards. As a result, serum testosterone level fluctuated with a significant decrease in the first and fifth weeks, a significant increase in the third week, and no significant difference in the second and fourth weeks. Serum E2 level decreased significantly throughout. Cyproterone acetate treatment (CA; 1 mg/fish daily for 21 days) from the second day of castration decreased the size and weight of the SV and the concentrations of total proteins, hexosamines, fructose and sialic acid. The antiandrogen treatment did not alter serum testosterone level but the E2 level was significantly decreased. It is concluded that the hypersecretory activity of the SV in castrates is a sequel to local synthesis and action of testosterone and the effect could be prevented by CA by blocking androgen actions.     

Importance of photoperiods in the regulation of ovarian activities in indian major carp Catla catla in an annual cycle

The present study attempted for the first time to explore the importance of photoperiod in the regulation of seasonal ovarian functions in any subtropical major carp. Adult Indian major carp Catla catla were transferred to a long photoperiod (LP; LD 16:8) or a short photoperiod (SP; LD 8:16) for 30 days on 4 dates corresponding to the beginnings of 4 reproductive phases in an annual cycle, and responsiveness of the ovary was evaluated by comparison with the gonadal weight (I(G)), relative number of developing oocytes, serum levels of vitellogenin, and the activity of 2 important steroidogenic enzymes, that is, Delta(5)3beta-hydroxysteroid dehydrogenase and 17.beta-hydroxysteroid dehydrogenase, in the ovary of fish in a natural photoperiod. Exposure of fish to LP during the preparatory phase (February-March) resulted in a significant (p < 0.001) increase in the values of vitellogenin and in the activity of both the steroidogenic enzymes but not in the ovarian weight and in the relative number of different stages of oocytes. A more stimulatory influence of LP was noted during the prespawning phase (April-May), when precocious maturation of ovary was evident from a significant (p < 0.001) rise in the values of each studied features of ovarian functions. However, no ovarian response was found when the fish were transferred to LP during the spawning (July-August) and the postspawning (September-October) phases. On the other hand, the SP was found to have an inhibitory influence on ovarian growth and maturation during the prespawning and the spawning phases or to have no influences on ovarian functions during the preparatory and the postspawning phases of an annual cycle. The results of our study provide the first evidence that photoperiod per se plays an important role in the seasonal maturation of ovary in a subtropical freshwater major carp.     

Structure of the ellipsoid sheath in the spleen of the armadillo (Dasypus novemcinctus). A light and electron microscopic study

A seasonal study of the seminal vesicles in relation to that of the testes had been conducted in the catfish, H. fossilis. The annual reproductive cycle of the catfish has been divided into (i) Preparatory period (February–April), (ii) Prespawning period (May–June), (iii) Spawning period (July–August) and (iv) Postspawning period (September–January). Testes exhibit initiation of spermatogenesis in the mid-preparatory period, but significant increase in weight of the testes accompanied by active spermatogenesis occurs during the prespawning period. In the spawning period, the testes are maximally enlarged and their seminiferous tubules are packed with spermatozoa. Following spawning, the testes gradually regress in the postspawning period. The seminal vesicles show initiation of secretory activity during the preparatory period but their recrudescence lags behind that of the testes by about a month. The seminal vesicles attain maximum weight and secretory activity during the spawning period. Thereafter, the seminal vesicles regress precipitously and sooner than the testes. The histochemical and biochemical studies on the seminal vesicles indicate that the secretion contains mucoproteins, acid mucopolysaccharides, primary proteoses, besides traces of phospholipids and native proteins.     

Hormonal mechanisms regulating hepatic vitellogenin synthesis in the gilthead sea bream,Sparus aurata

Experiments were carried out to study invitro the effects of 17-estradiol (E₂), homologouspituitary homogenate (HPH), and recombinant red sea bream growthhormone (sbGH) on vitellogenin (VTG) secretion from cultured sea breamliver fragments. Basal secretion of VTG was found to be significantlyhigher in the prespawning period, compared with sea bream liver in thespawning and postspawning periods. Similarly, the sea bream liverobtained during the prespawning period responded more significantly totreatments with E₂, HPH, or sbGH compared with sea breamliver during spawning. In the postspawning period, treatments withE₂, HPH, or sbGH were without significant effect on VTGsecretion level in sea bream liver. The level of E₂receptors was also analyzed by Western blot analysis. The resultdemonstrates a significantly higher level of E₂ receptors in the sea bream liver at the prespawning stage compared with those atthe spawning and postspawning stages. The findings support thehypothesis that homologous upregulation of estrogen receptors plays animportant role in the estrogen-sensitive control of VTG synthesis inthe sea bream liver.

     

Seasonal correlative changes between sex steroids and lipid levels in the freshwater female catfish, Heteropneustes fossilis (Bloch)

Circannual variation in plasma levels of testosterone (T), oestradiol-17β(E₂) and 17α-hydroxy-progesterone (17α-OHPg) were measured in female, Heteropneustes fossilis . T and E2 levels increased during the preparatory phase, reached their peak in the early prespawning phase and fell during the late prespawning phase to reach their lowest levels post-spawning. 17a-OHPg was detected from the late preparatory to the late spawning phase showing its peak during the early spawning phase. The levels of free fatty acids (FFA), monoglycerides (MG), diglycerides (DG), triglycerides (TG), phospholipids (PL), free cholesterol (CF)and esterified cholesterol (CE) were estimated in liver, plasma and ovaries. The preparatory phase showed hepatic lipogenic activity while during the prespawning phase TG lipolysis was increased by FFA. Ovarian CF was depleted by enhancing plasma E, levels during the prespawning phase. The gonadosomatic index reached its peak during spawning.     

Changes in populations of LHRH-immunopositive cell bodies following gonadectomy

One day after castration of male rats, plasma LH rose and the number of LHRH immunopositive neuronal perikarya decreased. As plasma LH continued to rise six days and three weeks post-castration, the number of LHRH immunopositive neurons also increased. The largest population of LHRH immunopositive neurons was detected three weeks post-castration and the cell group that showed the greatest increase was in the rostral preoptic area. In females, the largest population of LHRH immunopositive neurons was observed one day post-ovariectomy; at this time plasma LH levels were not significantly elevated above diestrous levels. Six days post-ovariectomy, LH levels were elevated and the number of LHRH immunopositive cells decreased. As LH levels continued to rise three weeks post-ovariectomy, the population increased in size. In males, primarily LHRH cells of the rostral preoptic area increased in in number; in females, the cell groups that increased were scattered over the diagonal band of Broca, preoptic and anterior hypothalamic areas. Although LHRH neurons demonstrated these variations following gonadectomy, there was no evidence of alteration(s) in molecular processing of precursor hormone.     

Seasonal cycles in total chemical composition of two Lake Baikal benthic-pelagic sculpins (Cottocomephorus, Cottoidei)

Annual cycles in chemical composition of the body, liver, gonads, red and white muscles were determined for two medium fat sculpins, Cottocomephorus grewingki and C. inermis , endemic to Lake Baikal. Their total lipid content ranged from 3 to 9% during the year. The content of defatted dry substance was 14–17%, similar to the protein content of the other Baikalian cottoid fishes. The prespawning period was characterized by a positive lipid and protein balance in both species. In C. grewingki the liver performed largely a metabolic function, whereas in C. inermis it served also as a storage site for lipid reserves. During spawning, total body lipids of females of both species showed a two- to threefold decrease. Males of C. grewingki guarding nests deplete greatly their total body lipids, which decrease seven- to ninefold by the time of larval hatching in comparison with the prespawning period, and constitute 1–2%. After spawning, lipid reserves of C. grewingki were quickly restored to the initial level (from about 3 to 9%) and maintained during the long sexual maturation period. In C. inermis total body lipids increased only twofold in comparison to those observed during the spawning period.     

Reproductive strategy and spawning activity of the European hake Merluccius merluccius (L.) in the Bay of Biscay

Histological examination of ovaries from European hake Merluccius merluccius from the Bay of Biscay sampled on a monthly basis from December 1996 to October 1997 demonstrated that European hake exhibit indeterminate fecundity. Oocyte development is asynchronous with a continuous oocyte size-frequency distribution in prespawning, spawning and postspawning females, and a gradual decrease in the mean diameter of advanced yolked oocytes as spawning proceeds. Spawning European hake were observed throughout the 11 month study period. The spawning fraction ranged from 0·085 to 0·207, which is equivalent to a batch interval of 5–12 days. The spawning fraction and proportion of spawning females were highest from January to March. Seasonal variation of atresia was inversely related to spawning fraction. Extensive atresia in individual fish was observed in September and October, when the spawning fraction was low.     

Inhibition of hCG-induced spawning by alpha-methylparatyrosine, a tyrosine hydroxylase inhibitor, in the catfish Heteropneustes fossilis (Bloch)

In this study, the effect of pharmacological inhibition of catecholaminergic activity on hCG-induced spawning was evaluated and correlated with tyrosine hydroxylase (TH) activity, the rate-limiting enzyme in catecholamine biosynthesis. Gravid female H. fossilis collected in both prespawning and spawning phases were given alpha-methylparatyrosine (alpha-MPT: 250 microg/g body weight, ip, an irreversible inhibitor of TH) and human chorionic gonadotropin (hCG: 100 IU/fish, ip) alone or in combination. The fish were sampled at different intervals for measuring hypothalamic and ovarian TH activity and checking spawning response. The administration of hCG resulted in ovulation and spawning in both phases with a higher response in the spawning phase. The administration of alpha-MPT did not induce any response, like the control fish. In the hCG + alpha-MPT groups, the spawning response of hCG was significantly inhibited and delayed by the inhibitor. The spawning response of hCG was accompanied by a significant increase in both hypothalamic and ovarian TH activity at 6 and 12 h of the injection. However, at 24hr the activity decreased except in the spawning phase. The alpha-MPT treatment inhibited TH activity significantly in a duration-dependent manner. In the hCG + alpha-MPT groups, enzyme activity was inhibited at all duration. The results indicate the involvement of catecholamines during the hCG-induced spawning and the specific functional nature of the involvement needs further investigation.     

Changes in trace metal concentrations in the tissues of the White Sea mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> over the reproductive cycle

Seasonal changes in the concentrations and total amounts (contents) of Cd, Zn, and Cu in the mussel Mytilus edulis cultivated in the White Sea were studied over the reproductive cycle (prespawning, spawning, and postspawning stages). The results, when compared with published data on the closely related species M. trossulus from the Sea of Japan, suggest that the seasonal dynamics of trace element contents in mussel tissues are related to specific geochemical conditions, as well as to the dynamics of changes in soft tissue weight over the reproductive cycle. The contents of Cd and Cu in the mussels before, during, and after spawning changed similarly in the mollusks from the White Sea and the Sea of Japan. Changes in the Zn content at different stages of the reproductive cycle of the White Sea mussels were similar to those in mussels from the Sea of Japan but had smaller amplitude. The concentration of Zn in the White Sea mussels was before spawning the highest, but still lower than in mussels from the Sea of Japan. After the spawning, the Zn concentration in the White Sea mussels, in contrast to the Pacific mussels, decreased because of the redistribution of this element during the prespawning period from the somatic tissue into the gonad.     

Acute effects of unilateral ovariectomy in the baboonPapio cynocephalus

The purpose of this study was to determine if steroids secreted by one ovary affected the steroid secretion of the other ovary by direct transportation of the steroids via uterine blood vessels. Either two or three baboons were scheduled for ovariectomy on the day of ovulation and on alternate days from 1–15 days before the expected day of ovulation. Two days before the scheduled ovariectomy, utero-ovarian vein blood from both sides was collected by the use of a laparoscope. Measurement of estradiol (E₂) was carried out in these samples. The ovary with a higher concentration of E₂ was designated the dominant side. At the time of ovariectomy utero-ovarian vein and uterine vein blood from the two sides was again collected. After removal of the dominant side ovary another sample of utero-ovarian and uterine vein blood was collected. The interval between removal of one ovary and blood collection from the contralateral side ranged from 2–15 min. Steroids E₂, progesterone (P), testosterone (T), and androstenedione (A) were measured in the blood plasma. Of the 21 baboons 12, 12, 11, and 10 baboons showed an increase in E₂, P, T, and A values, respectively, on the contralateral side after unilateral ovariectomy. The time elapsed between pre-ovariectomy blood collection and post-ovariectomy blood collection as well as the day of the follicular phase, when these samples were collected had no effect on the increases and decreases on the contralateral side. Statistical analysis, however, showed that the change in utero-ovarian vein or uterine vein hormone levels on the contralateral side after removal of one ovary was not significant for any of the four hormones E₂, P, T, and A. Thus there is no evidence to demonstrate cross circulation of steroids from one ovary to the other via direct vascular channels. This research was supported by NIH grant HD15300 toA. A. Shaikh.