Maturation of the hypothalamo-pituitary-gonadal axis of male Syrian hamsters.

Light-microscope immunocytochemistry (ICC) was used to investigate postnatal changes in the morphology of LHRH neurons in the brains of male Syrian hamsters and to relate these changes to more overt maturational developments within the hypothalamo-pituitary-gonadal axis. The animals were maintained under long-day photoperiods (14L:10D), and groups of 6-7 were killed at 10-day intervals from Day 15 to Day 65. Their brains were fixed with 4% paraformaldehyde, sectioned sagittally with a vibratome (75 microns), and processed for ICC using monoclonal LHRH antibody HU4H. Throughout the study period, the hamsters showed a progressive increase in plasma gonadotropin levels, closely followed by an increase in testicular weight and plasma testosterone levels. Histology of the testes revealed that spermatogenesis was already qualitatively completed by Day 35 and quantitative aspects were established by Day 45. Within the brain, LHRH neuronal perikarya were distributed primarily in the medial septal-preoptic area and the diagonal band of Broca; morphologically, these immunopositive neurons were either monopolar or bipolar. The total number of LHRH neurons detected in the areas examined was approximately 440 throughout the developmental period, and the relative proportions of monopolar and bipolar subtypes (86% and 14%, respectively) remained unchanged. In contrast, the area of the perikarya, as determined by autoimage analysis, showed a highly significant age-related increase, both for the monopolar and bipolar neurons. It is suggested that these developmental changes in the LHRH neurons reflect an increase in LHRH synthesis and may, therefore, provide a neuroendocrine trigger for the onset of puberty.     

Sustained reproductive responses in Djungarian hamsters (Phodopus sungorus) exposed to a single long day

Male and female Djungarian hamsters maintained from birth in a short photoperiod (8 h light per day; 8L:16D) showed substantial testicular and uterine growth in response to a single long photoperiod or a 15-min light pulse that interrupted the 16-h dark period at 18 days of age. These light regimens resulted in heavier testes and uteri at 30 and 35 days of age when compared with those of control animals. Similar results were obtained in hamsters maintained from birth to Day 18 in a long photoperiod (16L:8D), given a single longer day (20L:4D) or constant light on Day 18 and then transferred to a short photoperiod (8L:16D) on Day 19. At 35 days of age animals that received extended light treatment on Day 18 had significantly more developed reproductive structures than did control hamsters. The marked effects of brief light treatment in producing long-term changes in the reproductive axis provide a convenient mammalian model system in which to study neuroendocrine events that underlie photoperiodism.     

Social cues attenuate photoresponsiveness of the male reproductive system in Siberian hamsters (Phodopus sungorus)

Transfer of adult Siberian hamsters (Phodopus sungorus) from long (16 h light and 8 h dark, 16L:8D) to short (8L:16D) daily photoperiods induces an involution of the gonads and a cessation of reproductive behavior 8 to 10 weeks later. However, when male and female long-day hamsters were paired on transfer to short photoperiods, the males' gonads did not undergo the typical short-day response. Similarly, when male long-day hamsters were paired with refractory females (i.e., females housed in short photoperiods for at least 28 weeks so that they became unresponsive to short photoperiods), the response of the males' reproductive system to short photoperiods also was attenuated. Thus, social cues can override or delay the effects of photoperiod on the testes of this species. These results suggest that the inhibitory effects of long durations of melatonin secretion (in response to short photoperiods) on the male hypothalamic-pituitary-gonadal axis may be attenuated by social cues such as contact with the opposite sex.     

Influence of photoperiod and 6-methoxybenzoxazolinone on the reproductive axis of inbred LSH/Ss Lak male hamsters

Adult male Syrian hamsters of the inbred LSH/Ss Lak strain were maintained under a 14L:10D light cycle until 13 weeks of age. At this point, they were implanted s.c. with elastomer capsules that were either empty or packed with 30-40 mg of 6-methoxybenzoxazolinone (6-MBOA), a compound found naturally in some monocotyledonous plants; half of the animals from each treatment group were then kept in long days (14L:10D) or transferred to short days (9L:15D). Testicular size was measured and blood samples collected from each hamster immediately before capsule implantation and again 2, 4, 6 and 8 weeks later. Within just 2 weeks of exposure to short days the mean plasma levels of LH and FSH had significantly declined, in both the control and 6-MBOA-treated animals, and were basal within 4 weeks. Testicular size closely followed these gonadotrophin changes; within 4-6 weeks the testes from all of the short-day hamsters had completely regressed to a prepubertal size. At the end of the experiment, at Week 8, the animals were killed and various components of the hypothalamo-pituitary-testicular axis were compared between the treatment groups. The pituitary content of FSH and LH, testicular weight, mean serum level of testosterone, but not hypothalamic LHRH content or pituitary gland weight, were considerably lower in the short-day than in the long-day hamsters, regardless of whether or not they had been chronically treated with 6-MBOA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Termination of gonadal refractoriness in Turkish hamsters, Mesocricetus brandti

The Turkish hamster (Mesocricetus brandti) is a photoperiodic species. In this investigation, we characterized the photoperiodic requirements for termination of gonadal refractoriness, defined as the inability of the animal to respond to short-day treatment with gonadal regression. Paired testes weights were reduced to less than 20% of their original weight by 10 wk of 12L:12D treatment. This was followed by spontaneous testicular recrudescence (completed by Week 25 of 12L:12D treatment), the overt indication of refractoriness to short photoperiods. Next, the period of long-day exposure sufficient for termination of refractoriness was determined. Refractory males were exposed to 16L:8D for 5 to 20 wk. Ten weeks of 16L:8D treatment was enough for the animals to regain the sensitivity to a second challenge of 12L:12D treatment. Fifteen weeks of 20L:4D or 16L:8D terminated refractoriness in female Turkish hamsters; 20L:4D therefore was not interpreted as a short day by refractory hamsters. This was unexpected because in photosensitive animals this photoperiod acts like a short day, causing gonadal regression. These results suggest that Turkish hamsters are similar to Syrian hamsters in that both species require two or more months of long days in summer to recover sensitivity to the short days of the following fall.     

Neuroendocrine changes in male hamsters following photostimulation

Transfer of gonadally regressed male golden hamsters from a short (5 L:19 D) to a stimulatory (14 L:10 D) photoperiod elicits, within 24 hr, significant changes in hypothalamic dopamine, serotonin, and possibly norepinephrine metabolism. Hypothalamic LHRH content was significantly elevated in short-photoperiod animals, but within 24 hr of transfer to a 14:10 photoperiod, LHRH declined to levels not different from those in hamsters maintained continuously in a long photoperiod. Plasma FSH levels were also significantly elevated within 24 hr of transfer, but increases in plasma LH were somewhat slower. Chronic treatment with the tyrosine hydroxylase inhibitor, alpha-methyl tyrosine (alpha MPT), which inhibits catecholamine synthesis, blocked the effect of a stimulatory photoperiod on plasma FSH levels, while treatment of 5:19 hamsters with the catecholamine precursor, L-dopa, mimicked the effects of photostimulation on plasma FSH levels. Testicular weights were not affected by alpha MPT or L-dopa treatment for 1 week. From these data, it appears that endocrine events associated with photoperiod-induced testicular recrudescence are under the control of hypothalamic neurotransmitters.     

Chronic exposure to short photoperiod inhibits free thyroxine index and plasma levels of TSH, T4, triiodothyronine (T3) and cholesterol in female Syrian hamsters

1. Chronic exposure of female Syrian hamsters (Mesocricetus auratus) for 9 weeks to a short photoperiod (10L:14D) depressed the pituitary-thyroid axis as indicated by a drop in circulating titers of thyroid stimulating hormone (TSH), thyroxine (T4), triiodothyronine (T3) and the free thyroxine index (FT4I) compared to animals maintained under long photoperiodic conditions (14L:10D). 2. Short day treatment also reduced plasma cholesterol levels. 3. Neither plasma triglycerides, glucose nor growth hormone (GH) levels differed between hamsters exposed to short or long daily photoperiods.     

Melatonin mediates alternation of seasonality in Syrian hamsters

The annual cycle of reproductive activity in the Syrian hamster, Mesocricetus auratus, is the result of interaction between seasonal changes in daylength (photoperiodism) and seasonal changes in responsiveness to daylength (seasonality). The present experiment was designed to investigate the role of the pineal gland and its hormone, melatonin, in the alternation of seasonality (scotosensitivity and scotorefractoriness). Male hamsters were maintained on short daylengths (10L:14D) to establish scotorefractoriness, and then they were transferred to long daylengths (14L:10D) for conversion to scotosensitivity (sensitive to short daylengths). Before transfer to long daylengths, some of the hamsters were pinealectomized and others were sham-operated or unoperated. Some of the pinealectomized hamsters received single daily melatonin or saline injections while on long daylengths. After 14 wk on long daylengths, the hamsters were transferred to short daylengths for 10 wk to test for conversion to scotosensitivity. Pinealectomized hamsters were given three daily melatonin injections while on short daylengths. Such treatment is known to promote gonadal regression in scotosensitive but not in scotorefractory hamsters. Examination of testes after the short daylength interval revealed that exposure of nonpinealectomized hamsters to long daylengths had reestablished scotosensitivity (regressed testes). Pinealectomized hamsters that received no melatonin replacement while on long daylengths remained scotorefractory (enlarged testes), whereas those that received single daily injections of melatonin during long daylengths were found to be scotosensitive. These results indicate that a daily pulse of melatonin during expsoure to long daylengths has an important role in reestablishing responsiveness (scotosensitivity) to short daylengths.     

Effects of short photoperiod on the ability of golden hamster pituitaries to secrete prolactin and gonadotropins in vitro

Transfer of male golden (Syrian) hamsters from a 14L:10D (light:dark) to a 5L:19D photoperiod induced significant changes in pituitary function tested in vitro. Within 27 days after transfer to a 5L:19D photoperiod, basal prolactin (Prl) release was significantly depressed and response to dopamine (DA) was significantly enhanced as compared to Prl release by pituitaries from 14L: 10D hamsters. Follicle-stimulating hormone (FSH) release tended to be depressed after 9 or 27 days of 5L:19D exposure, but the effect was not significant. After 77 days of 5L:19D exposure, Prl release was further suppressed, while FSH release surpassed that seen in 14L:10D pituitaries. In vitro FSH response to luteinizing hormone releasing hormone (LHRH) was also enhanced at this time. After 15 weeks of exposure to a short photoperiod, FSH secretion was still elevated above control levels, but Prl release and Prl response to DA were no longer different from that of 14L: 10D controls. Secretion of luteinizing hormone (LH) in vitro, either basal or LHRH stimulated, was not affected by photoperiod at any time tested. From these results, we conclude that short photoperiod exposure does not reduce the pituitary's ability to secrete LH or FSH, although secretion of Prl is severely attenuated.     

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.     

Early photoperiod history and short-day responsiveness in Siberian hamsters

Siberian hamsters exhibit seasonal, photoperiod influenced cycles of reproductive activity, body size, pelage characteristics, and thermoregulatory behavior. Laboratory populations generally exhibit inter-individual variability in expression of photoperiod responsiveness, with a subset of individuals that fail to show the species typical responses to short photoperiod. This variability is partly explained by a genetic component, as it has been possible to increase the number of short-day nonresponders by artificial selection. Responsiveness to short photoperiod is also substantially influenced by photoperiod history in this species; hamsters that have been raised under long (16L) or very long (18L) day lengths are less likely to exhibit winter-type responses to short days as compared to hamsters raised under an intermediate (14L) day length. In the present experiment, we examined effects of age and early photoperiod history in a strain of Siberian hamsters that had been selected for short-day nonresponsiveness. Hamsters transferred into short photoperiod on the day of birth were uniform in exhibiting winter-type responses. However, hamsters raised until 25 days of age in either continuous illumination or in 16L exhibited variation in responsiveness when subsequently moved into short photoperiod. We conclude that virtually all hamsters of the short-day nonresponsive strain are born responsive to short days. Subsequent development of resistance to potential short day effects is dependent on age and/or photoperiod history.     

Effect of photoperiod on the size of the Leydig cell population and the rate of recruitment of Leydig cells in adult Syrian hamsters

The number of Leydig cells was determined by stereologic procedures in adult Syrian hamsters housed in long days (14L:10D) to maintain testicular activity (active), in short days (5L:19D) for 12-13 wk to induce testicular regression (photoperiod-induced regressed), or in short days for a period of 21 wk or more to allow spontaneous gonadal recrudescence (spontaneously recrudesced). Testes were removed, sliced, fixed, embedded in Epon 812, and observed by bright-field microscopy. Testicular and seminal vesicle weights, plasma testosterone concentration, total Leydig cell volume per testis, and volume of single Leydig cell were greater (p less than 0.01) in active and recrudesced animals than in regressed animals. The density of Leydig cells was greater in the regressed testes, but the total number per testis was not influenced by photoperiod. In Experiment 2, the rate of recruitment of Leydig cells was determined in 5 adult hamsters exposed to long days (active) or 5 hamsters whose testes were regressed by exposure of animals to short days for 13 wk followed by long-day exposure to initiate testicular growth (photoperiod-induced recrudescing). Hamsters were injected for 3 days/wk for 3 wk with tritiated thymidine, 0.5 or 1 microCi/g body weight. Testes were fixed and tissues prepared, as above, and processed for autoradiography. Again, the photoperiod did not influence the number of Leydig cells per testis. Labeling of Leydig cell nuclei revealed that recruitment of new Leydig cells occurred at approximately 1.3% per day in recrudescing testes but also occurred at approximately 0.6% per day in active testes. Without change in the total number of Leydig cells, new Leydig cells were added continually to the existing population in adult hamsters with either recrudescing or active testes.     

Effects of short-term treatment with testosterone on the secretion of FSH and LH in castrated golden hamsters exposed to short days

Castrated hamsters which were transferred from long (14L:10D) to short (9L:15D) days and received testosterone-filled capsules for 1 week after transfer failed to show a significant suppression in the plasma levels of FSH and LH after capsule removal. In contrast, gonadotrophin concentrations were suppressed in hamsters in which the long-day castration response had been blocked with exogenous testosterone. After castration on long days and exposure to 10 weeks of short days pituitary gland weight and gonadotrophin content, as well as plasma FSH titres, were higher in control animals than in those that had received testosterone implants for 7 weeks of short days. The results suggest that failure of castrated hamsters to respond to the suppressive effects of short days reflects castration-induced changes in hypothalamo-pituitary physiology rather than a neuroendocrine mechanism by which photoperiod modulates gonadotrophin secretion.     

Testicular development in Siberian hamsters depends on frequency and pattern of melatonin signals

We investigated the impact of frequency and pattern of melatonin signals on reproductive development in Siberian hamsters. Juvenile males gestated in short day lengths and housed in constant illumination to suppress melatonin secretion were infused with melatonin for 5 h either once or twice per day for 20 days. Melatonin infusions at either frequency produced equivalent increases in testes and body weights that exceeded those of animals infused with saline but were indistinguishable from those of hamsters transferred to long day lengths. The reproductive system appears to be maximally stimulated by a single short melatonin signal each day. Other animals kept from birth in a short photoperiod were treated 6 h after onset of darkness with the beta-adrenergic receptor antagonist DL-propranolol to shorten melatonin secretion on the night of injection but not on subsequent nights. This permitted interpolation of short nightly melatonin signals of 4-5 h duration against a background of long melatonin signals of 10-12 h duration on other nights. Treatment regimes that maintained a 1:1 ratio of short to long melatonin signals for 8 wk stimulated reproductive development; a 1:2 signal ratio, in each of three different patterns, was uniformly ineffective. The number of successive short melatonin signals had little influence on the interval across which successive melatonin signals were summated to influence photoperiodic traits. The neuroendocrine axis appears more responsive to short melatonin signal frequency than pattern for development of the summer phenotype.     

Elimination of short-day melatonin signaling accelerates gonadal recrudescence but does not break refractoriness in male Turkish hamsters

Long days stimulate and short days (SDs) inhibit the reproductive axis of photoperiodic rodents. In long-day Turkish hamsters, unlike most other rodents, elimination of pineal melatonin secretion by constant light or pinealectomy initiates a cycle of gonadal involution and recrudescence outwardly similar to that induced by short days. The present study assessed whether short days and constant light induce the seasonal reproductive cycle via common or different interval timing mechanisms. Male hamsters that had undergone gonadal involution in SDs for 8 or 14 weeks were treated with LL for 14 and 8 weeks, respectively. If SDs and LL act via independent mechanisms, then gonadal quiescence of SD-regressed males, which normally lasts 10 weeks, might be extended by LL treatment; alternatively, if SDs and LL act on the same timer, or the timer cannot be retriggered, then LL will not extend the duration of reproductive quiescence. Neither of these outcomes materialized. Instead, male hamsters exposed to LL while reproductively quiescent exhibited accelerated gonadal recrudescence. Extended LL treatment did not restore responsiveness to SDs in photorefractory hamsters. In Turkish hamsters, photoperiodic history determines whether constant light inhibits or stimulates the hypothalamic-pituitary-testicular axis.     

Short day lengths delay reproductive aging

Caloric restriction and hormone treatment delay reproductive senescence in female mammals, but a natural model of decelerated reproductive aging does not presently exist. In addition to describing such a model, this study shows that an abiotic signal (photoperiod) can induce physiological changes that slow senescence. Relative to animals born in April, rodents born in September delay their first reproductive effort by up to 7 mo, at which age reduced fertility is expected. We tested the hypothesis that the shorter day lengths experienced by late-born Siberian hamsters ameliorate the reproductive decline associated with advancing age. Short-day females (10L:14D) achieved puberty at a much later age than long-day animals (14L:10D) and had twice as many ovarian primordial follicles. At 10 mo of age, 86% of females previously maintained in short day lengths produced litters, compared with 58% of their long day counterparts. Changes in pineal gland production of melatonin appear to mediate the effects of day length on reproductive aging; only 30% of pinealectomized females housed in short days produced litters. Exposure to short days induces substantial decreases in voluntary food intake and body mass, reduced ovarian estradiol secretion, and enhanced production of melatonin. One or more of these changes may account for the protective effect of short day lengths on female reproduction. In delaying reproductive senescence, the decrease in day length after the summer solstice is of presumed adaptive significance for offspring born late in the breeding season that first breed at an advanced chronological age.     

Localization of luteinizing hormone-releasing hormone in the forebrain of the pig

The distribution of luteinizing hormone-releasing hormone (LHRH)-immunostained perikarya and processes was examined in the forebrains of six sexually mature female pigs by use of indirect biotin-avidin horseradish peroxidase immunocytochemistry. Two primary antisera (Drs. Y.F. Chen and V.D. Ramirez CRR11B73 and Miles-Yeda UZ-4) yielded positive staining. Adjacent sections treated either primary antiserum preabsorbed with LHRH or with normal rabbit serum substituted for primary antiserum lacked positive staining. The greatest proportion of LHRH-immunostained perikarya were found in the medial preoptic area adjacent to the organum vasculosum of the lamina terminalis. The LHRH-immunostained perikarya were also scattered rostrally in the diagonal band of Broca, and within the lateral hypothalamic area, paraventricular nucleus, periventricular zone, suprachiasmatic nucleus, and medial basal hypothalamus. LHRH-immunostained processes, which extended from the medial preoptic area, coursed either along the ventral surface to the median eminence or medially and ventrally along the third ventricular wall ventrally to the median eminence and caudally to the level of the mammillary bodies. Extrahypothalamic processes were located adjacent to the lateral ventricular floor and the third ventricle from the lateral septal area (stria terminalis) to the level of the habenular nucleus. LHRH-immunostained neurons were unipolar, bipolar, and multipolar. Close associations between individual LHRH-immunostained neurons were observed.     

Role of the pineal and its hormone melatonin in the termination of photorefractoriness in golden hamsters

Continuous exposure of male hamsters to short day lengths induces testicular regression. This is followed many weeks later by spontaneous recrudescence of the testes with reinitiation of spermatogenesis and function of the accessory sexual glands. Hamsters at this stage of the annual reproductive cycle are refractory to short photoperiods--even continuous darkness will not induce another bout of testicular regression. Animals refractory to short days are also refractory to the pineal hormone melatonin and a number of investigators attribute spontaneous recrudescence and photo and melatonin refractoriness to a developed target cell insensitivity to endogenous melatonin from the pineal. Refractoriness is terminated by exposure to long days for at least 11 weeks. The pineal gland is reported to be essential for this process. We report here the effects of pinealectomy, daily melatonin injections, and constant-release melatonin implants on the ability of male hamsters to recover from the refractory state. In the absence of the pineal gland, refractory male hamsters did not discriminate (count?) 15 weeks of long days to terminate refractoriness. Daily melatonin injections at 1900 h, but not at 1200 h (lights 0600-2000 h) during the 15 weeks of long-day exposure blocked the recovery from refractoriness. Constant-release melatonin implants abolished the animals ability to measure 12 and 15 weeks of long days to terminate refractoriness. These results demonstrate that general target tissue insensitivity to melatonin cannot account for the refractory state in hamsters, that a multiplicity of target tissues may exist for melatonin to account for its varied roles throughout the annual reproductive cycle in hamsters, and that the pineal gland is intimately involved in the animals' ability to measure a prescribed duration of long days to terminate refractoriness.     

Leptin inhibits the reproductive axis in adult male Syrian hamsters exposed to long and short photoperiod

The aim of the study was to investigate the effects of acute leptin treatment of adult Syrian hamsters exposed to a long (LP, eugonadal males) and short photoperiod (SP, hypogonadal males). Animals were exposed to LP (L:D 14:10) or SP (L:D 10:14) for 10 weeks. Afterwards, both LP and SP hamsters were allocated to a control (SP-C, LP-C) or leptin-treated group (SP 3, SP 10, SP 30 or LP3, LP 10, LP 30). One hour before sacrifice, a single dose of leptin (3, 10 or 30 μg/kg) or vehicle was administered (i.p.) to the males. Testis weight, serum and pituitary luteinizing hormone (LH) concentrations, as well as the hypothalamic concentration of gonadotropin-releasing hormone (GnRH) were recorded. Histological analysis of the testis was performed and GnRH concentration in the culture medium of hypothalamic explants was examined. A dramatic regression of testicular weight and histological atrophy of seminiferous tubules, as well as a decrease in serum and pituitary LH concentrations were found in SP males. All doses of leptin significantly reduced serum LH levels and medium GnRH concentrations in both photoperiod groups. Pituitary LH and hypothalamic GnRH concentrations were not affected by leptin. In conclusion, we demonstrated that leptin inhibited the reproductive axis of Syrian male hamsters exposed to LP and SP and fed ad libitum.     

Hypothalamic alterations and reproductive aging in female rats: evidence of altered luteinizing hormone-releasing hormone neuronal function

Prior to the age-related loss of regular estrous cycles, female rats exhibit an attenuated preovulatory LH surge, a sign that reproductive decline is imminent. Numerous studies have revealed an important role for the hypothalamus in aging of the reproductive axis in this species. Because LHRH represents the primary hypothalamic signal that regulates gonadotropin release, assessments of LHRH neuronal activity can provide a window into hypothalamic function during reproductive aging. Studies of the dynamic activity of LHRH neurons during times of enhanced secretion have revealed deficits in middle-aged females. Available data are consistent with a decline in LHRH synthesis, transport, and secretion in middle-aged females during times of increased demand for LHRH output. Moreover, the alterations noted in LHRH neuronal function could account, in part, for the attenuation and eventual loss of the preovulatory LH surge with age. Elements extrinsic to LHRH neurons undoubtedly contribute to the decline in the parameters of LHRH neuronal function observed in middle-aged females. Whether alterations intrinsic to LHRH neurons also play a role in the age-associated reduction in LHRH synthesis and secretion remains to be determined. Recent examinations of hormone profiles during the perimenopausal period suggest that a potential hypothalamic contribution to aging of the reproductive axis in women warrants further examination.