Effect of photoperiod on the distribution patterns of androgen receptors and steroid hormone concentrations in ovaries of bank voles

Cellular distribution patterns of the androgen receptor in ovaries of female bank voles, born and reared in long (18:6; LD) or short (6:18; SD) photoperiods, have been studied to understand effects of androgens in female gonads. The photoperiod is one of the most important factors in bank voles, which are seasonal breeders, that regulates both morphology and hormonal function of the ovary. Androgen receptors were visualized immunocytochemically, using a specific monoclonal antibody against androgen receptor protein. LD ovaries contained more follicles and showed a different androgen receptor distribution pattern than SD ovaries. In LD ovaries, androgen receptors were strongly expressed in granulosa cells of primordial, primary, and preantral follicles as well as in theca and stromal cells. Positivity was moderate and limited to antral and cumulus regions in large follicles of LD ovaries. In contrast, androgen receptor immunopositivity was intense in the granulosa layer, theca and interstitial cells of large follicles of SD ovaries. A novel observation was the very intense immunostaining of oocyte cytoplasm in primordial and primary unilaminar follicles of LD ovaries. It can be concluded that the androgen receptor is involved in the maturation of oocytes.     

Steroid levels and the spatiotemporal expression of steroidogenic enzymes and androgen receptor in developing ovaries of immature rats

Immunoexpression of 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450c17 (P450c17), androgen receptor (AR), and steroid contents were studied in the ovaries of immature female Wistar rats killed between postnatal days 1 and 30. During days 1-7, ovarian somatic structures lacked AR, 3β-HSD and P450c17, except for the surface epithelium, which featured the presence of these three proteins, suggestive of its androgen responsiveness and steroidogenic function. On day 10, AR appeared in many somatic structures, including the granulosa layers, which coincided with the P450c17 immunoexpression in some theca/interstitial cells, and an increase in ovarian androgen concentration. On the following days a further rise in ovarian androgen and progesterone contents paralleled an increase in 3β-HSD and P450c17 immunoexpression in the theca layer cells and primary interstitial cells. However, the development of the follicles constituting the first follicular wave was aberrant, since they lacked AR expression until the preantral stage and were characterized by a delayed onset and much lower expression of the thecal P450c17. They could not ovulate, since ovarian content of estradiol was too low to evoke the LH surge. The clusters of the secondary interstitial cells found on day 30 exhibited predominant expression of 3β-HSD over P450c17, suggesting more intensive progesterone than androgen synthesis in these structures.     

缝隙连接蛋白43和黄体生成素受体在小鼠卵巢中的表达

目的 观察小鼠卵泡发育过程中缝隙连接蛋白43（Cx43）和黄体生成素受体（LHR）在小鼠卵巢中的表达以及黄体生成素(LH)对卵巢Cx43表达的影响,为这两种分子与卵泡发育的关系提供实验依据。 方法 选择出生后1d、4d、7d、2周、3周、4周、6周、8周龄雌性C57小鼠共8组,每组10只,取卵巢。HE染色观察卵巢形态、卵泡发育并测量颗粒细胞体积分数。免疫组织化学和Western blotting观察卵巢组织中Cx43及LHR的表达,Western blotting检测不同浓度黄体生成素（LH）孵育后对卵巢Cx43表达的影响。 结果 HE染色显示,出生1d小鼠卵巢中见原始卵泡,出生7d卵巢内出现初级卵泡,2周时出现窦前卵泡和少量窦卵泡,3周、4周时,出现较多大的窦卵泡,6～8周时出现黄体。卵泡颗粒细胞体积分数在2周后的卵巢中明显增加。Cx43表达在各阶段卵泡的颗粒细胞胞膜上,LHR表达在卵泡膜细胞、颗粒细胞及卵母细胞胞质中。Cx43和LHR出生后2周卵巢中的表达开始明显增加,并随卵泡发育表达逐渐增强,在3周、4周、6周和8周维持相对较高水平。LH体外干预可增加卵巢组织Cx43的表达。 结论 Cx43和LHR在卵泡发育中发挥重要作用,LH可能通过LHR促进卵巢卵泡颗粒细胞Cx43的表达。     

Short day lengths decrease body mass of overweight female meadow voles

Groups of adult ovariectomized (ovx) voles and sham-ovx controls were placed in a short photoperiod or left in the original long photoperiod (10 or 14 hr light/day). In the next 12 weeks long-day ovx voles increased their body mass by 11.3%, whereas animals transferred to the short-day photoperiod manifested an 11.9% decline in body mass. Short photoperiods not only reversed pre-existing weight gains attributable to ovariectomy, but also prevented further increases in body mass associated with the withdrawal of ovarian hormones. Fat-free dry carcass mass was increased by ovariectomy and decreased by short photoperiods; lipoprotein lipase activity of white adipose tissue was unaffected by ovariectomy or photoperiod. We suggest that reductions in body mass in voles transferred to the short photoperiod are mediated by changes in pineal melatonin secretion.     

淫羊藿甙腹腔注射对新生大鼠卵巢发育的影响

目的应用淫羊藿甙对新生大鼠进行腹腔注射,了解其对卵泡发育和卵母细胞凋亡的影响。方法用淫羊藿甙对出生后1～8d大鼠腹腔注射50mg/（kg·d）,分别取出生后2、4、8d龄卵巢,用苏木素-伊红（hematoxylin-eoxin,HE）染色观察不同发育阶段卵泡比例,末端脱氧核苷酰基转移酶介导性dUTP切口末端标记（TdT-mediated dUTP Nick-End Labeling,TUNEL）荧光染色检测卵巢内卵母细胞凋亡变化。结果在淫羊藿甙腹腔注射组的新生的鼠中,1、2d龄卵巢内未装配卵泡比例及4、8d龄卵巢内原始卵泡比例均高于对照组;各日龄组卵巢中卵母细胞TUNEL阳性率明显低于对照组。结论淫羊藿甙可能通过延缓卵母细胞巢破裂,抑制原始卵泡的发育启动,减少卵母细胞凋亡从而增加新生大鼠卵巢中卵母细胞的储备量。     

Immunohistochemical localization of androgen receptor in the human ovary throughout the menstrual cycle in relation to oestrogen and progesterone receptor expression.

In order to elucidate the role of androgen receptors (AR) in human ovaries, we examined their immunohistochemical localization, in comparison with oestrogen receptors (ER) and progesterone receptors (PR), at various stages of the menstrual cycle and follicular development. Primordial and primary follicles did not express AR. In granulosa and thecal cells of secondary follicles there was weak nuclear staining for AR. Granulosa cells of dominant follicles showed moderate nuclear staining for AR, which was stronger than that in thecal cells. In the luteal phase, the staining intensity for AR was strongest in the early luteal phase just after ovulation and declined gradually thereafter. Thecal cells of atretic follicles showed moderate nuclear staining for AR, which was a little stronger than that in dominant follicles. There was weak nuclear staining for AR in stromal cells surrounding follicles. Though there was variation in the staining intensity, AR were present at almost all stages of the menstrual cycle. There is a possibility that androgens, mediated by AR, may play an essential role in follicular growth and maturation, atresia and luteinization as autocrine or paracrine agents.     

The female-to-male transsexual patient: a source of human ovarian cortical tissue for experimental use

We demonstrated that ovaries removed from female-to-male transsexuals can be used as research material for ovarian cryopreservation, grafting and culture studies. A 21 year old female-to-male transsexual individual, who had been treated with androgens for 12 months, donated her ovaries removed in the sex reversal operation. She had normal numbers of, and proportions of, primordial (98.6%) and primary (1.4%) follicles in her ovarian cortex. After freezing and thawing the ovarian tissue was grafted to immuno-incompetent mice, which were stimulated by human recombinant FSH for 14 days, 10 weeks after transfer. Initiation of growth had occurred in the grafts in which only 79.4% of follicles were primordial, 17.1% primary, and in which there were also secondary and pre-antral follicles. Because long-term androgen treatment does not appear to cause depletion of the primordial follicle pool or affect the developmental capacity of the follicles, ovaries from individuals who undergo sex-reversal operations are an excellent source of tissue for research and maybe even for oocyte donation in the future.     

丹参注射液对冻融小鼠卵巢移植早期光镜结构及超微结构的影响

目的 探讨丹参注射液对冻融小鼠卵巢同种异体移植早期光镜结构及超微结构的影响.方法 收集1日龄小鼠卵巢,慢冻速融后移植至F1代8～12周雄鼠的肾被膜下,分别于移植后第2天(2d)、第7天(7d)和第14天(14d)回收两组卵巢移植物.光镜和电镜技术观察卵巢组织冻融前后和移植前后的组织形态学改变、计数卵泡数量.结果 小鼠卵巢经冻融后原始卵泡的存活率为87.9%.光、电镜观察卵巢组织移植后7d卵泡细胞和颗粒细胞结构欠完整,移植后14d卵泡结构恢复正常.移植后14d丹参组各级卵泡数及卵泡存活率(30.1%)多于生理盐水组(13.2%),差异显著(P＜0.05).结论 结合光镜和电镜观察能更全面地评价冻融过程和移植过程对小鼠卵巢组织的影响,移植早期的缺血缺氧对小鼠卵巢组织造成了远比冻融过程更严重的损伤;丹参注射液可能促进移植后冻融小鼠卵泡发育,减少细胞凋亡.     

The effect of ovarian steroids and photoperiod on body fat stores and uncoupling protein 2 in the marsupial Sminthopsis crassicaudata

To determine the effects of photoperiod and ovarian steroids on fat stores in the marsupial S. crassicaudata, animals were ovariectomised (OVX) or sham operated, and maintained under either short-day (SD) or long-day (LD) photoperiods for 104 days. Photoperiod had no effect on body weight in the sham animals. In the LD OVX animals, body weight fell and remained below baseline for about 45 days, whereafter it returned to baseline. In contrast, body weight of SD OVX animals increased over the first 45 days then returned to baseline. Tail width (a reflection of body fat stores) increased in both sham and OVX animals exposed to SD. When exposed to LD, tail width increased only in the OVX animals. There was no effect of either photoperiod or OVX on total cumulative energy intake. Leptin mRNA expression was increased in the LD OVX animals compared to the shams. Photoperiod had no effect on UCP2 mRNA expression in any tissue; however, OVX decreased UCP2 mRNA expression in muscle. These data indicate that in S. crassicaudata: (a) fat mass increases in response to both SD photoperiod and OVX and they have additive effects; (b) the effects of photoperiod on fat mass are mediated by both gonadal steroid dependent and independent mechanisms; (c) alterations in UCP2 mRNA expression may mediate the effect of OVX, but not photoperiod; and (d) UCP2 mRNA is differentially regulated in muscle and fat.     

Histopathological changes in androgenized ovaries are recovered by melatonin treatment

Nandrolone decanoate (ND) is a synthetic steroid, which promotes adverse effects on the ovarian tissue, and melatonin (MLT) exhibits a number of beneficial properties in the reproductive system. This study evaluated the general features of the ovarian tissue and the immunoexpression of sex steroid receptors in ND‐treated rats that were submitted to short‐term melatonin treatment. Adult rats received mineral oil (control group) and ND at doses of 7.5 mg/kg for 15 days (ND‐treated group). The treatment with MLT (10mg/kg for 7 days) was given alone, before or in combination with ND. All ND‐treated animals showed persistent dioestrus. In the androgenized groups that received MLT, ovarian morphology and size, and the number/area of corpora lutea were recovered. The number of healthy and atretic follicles was recovered when MLT was administered prior to ND; this was similar to the ovaries of control and MLT groups. There was a decrease in estrogen receptors immunostaining in the follicles of androgenized rats that were treated with MLT, and pretreatment with MLT reduced the expression of androgen receptor in atretic follicles and corpora lutea, when compared with ND‐treated group. We conclude that MLT treatment recovered the histopathological aspects of the androgenized ovaries, and MLT pretreatment was the most effective.     

Female meadow voles housed in long and short daylengths respond to exogenous estrogen with similar mating latencies

After weaning, adult female meadow voles were maintained for 7 weeks in either long (LD, 14 h light/day) or short photoperiods (SD, 10 h light/day). They were then ovariectomized and implanted with 3-week, timed-release estrogen pellets (0.0, 0.001, 0.05, or 0.5 mg/pellet of 17-beta-estradiol). An additional group received a sham ovariectomy (intact) and a 0.0-mg/pellet control (no estrogen) pellet. One week after surgery, females were paired with an LD sexually experienced male. Each pair was videotaped continuously until the first intromission or for 2 weeks. LD sham animals mated significantly earlier than did SD sham animals (P=.05). However, there were no differences in mating latencies between LD and SD control groups or between any of the LD and SD groups receiving estrogen replacement (P>.05). In addition, no ovariectomized animals receiving either the control or the 0.001-mg/pellet estrogen dosage mated within the 2-week time period, while all shams and all animals receiving either the 0.05- or 0.5-mg/pellet doses mated. The results indicate that there is some minimal amount of estrogen that is necessary for meadow voles to enter behavioral estrus and that LD and SD females do not differ in their sensitivity to estrogen, since animals in both photoperiods mate with similar latencies as long as enough estrogen is present.     

Graafian follicles are cooler than neighbouring ovarian tissues and deep rectal temperatures

In order to establish appropriate culture temperatures for in-vitro maturation of pig ovarian oocytes, large Graafian follicles (7-10 mm diameter) were sensed by infra-red technology during the latter part of a spontaneous oestrous cycle. Temperatures were measured under systemic anaesthesia almost instantaneously upon revealing the ovaries at mid- ventral laparotomy. Temperature differentials were observed within all 16 ovaries sensed in 14 animals. Ovaries were always cooler than deep rectal temperatures (mean rectal temperature was 38.0 +/- 0.4 degrees C; range 37.5-38.6 degrees C) and mature follicles always cooler than ovarian stroma (35.6 +/- 0.3 degrees C versus 37.3 +/- 0.2 degrees C respectively; P < 0.01). Such follicles were frequently 1.5-1.8 degrees C cooler than the adjacent stroma, the mean being 1.7 +/- 0.4 degrees C. Small Graafian follicles (< 5-6 mm diameter) and recent ovulations did not show this differential. The control experiment of excising an ovary, deep freezing it in liquid nitrogen, and then restoring it to the body cavity before further sensing indicated that intra-ovarian temperature gradients depended on the activity of living tissues and/or a functional blood supply. Furthermore, calculation of anticipated rates of cooling for exposed Graafian follicles strongly suggested that artefacts could not have been solely responsible for the observed temperatures. Endothermic reactions within mature follicles were thus brought into focus. It is concluded that follicular temperatures may influence the meiotic progression and cytoplasmic maturation of oocytes and act to regulate enzymatic activity in the biosynthetic pathways for steroid and/or peptide hormones.      

Photoperiodic regulation of body mass, food intake, and reproduction in meadow voles

Adult male voles were maintained for 10 wk in long or short photoperiods (14 or 10 h of light/day). A third group of animals housed in the long photoperiod was implanted with capsules containing melatonin. Body weight and food intake were measured weekly; various tissues were weighed and analyzed at the time of autopsy. After 10 wk, voles in the short photoperiod weighed 20% less and consumed 30% less food than those housed in the long photoperiod. Total body water and lean body mass were reduced in the short-day animals, although the size of the brown adipose tissue was not affected. White adipose tissue lipoprotein lipase (LPL) activity was markedly reduced in the short-day voles who also manifested gonadal regression and suppression of spermatogenesis. Melatonin mimicked the effects of short photoperiods on LPL activity and on lean body mass; other parameters for melatonin-treated animals were intermediate between those of untreated long- and short-day voles. We hypothesize that winter weight losses experienced by meadow voles in the field are mediated by decreases in the duration of the daily photophase and that the reduction in body mass permits overwintering voles to reduce their energy requirements and the amount of time devoted to foraging. At least part of the seasonal decline in body mass appears due to a decrease in gonadal hormone secretion.     

Comparison of follicle steroidogenesis from normal and polycystic ovaries in women undergoing IVF: relationship between steroid concentrations, follicle size, oocyte quality and fecundability

Studies of ovarian stimulation for IVF have suggested a relationship between follicle size and pregnancy rates. Furthermore the follicular endocrine environment is correlated with oocyte quality. The aim of this study was first to verify the relationship between follicular steroid content, follicular size, oocyte maturity and fertilization outcome in women with normal ovaries following recombinant human FSH (rhFSH). Secondly this study was extended to women with polycystic ovarian syndrome (PCOS). Fifty-nine patients (31 normal, 28 PCOS) underwent conventional IVF with rhFSH induction. Follicular diameter was classified as small (8-13 mm) or large (>14 mm) and sex steroid content was analysed for each group. Oocyte maturity was studied according to nuclear maturation the day after fertilization. In both ovulation groups, 17 beta-oestradiol and progesterone concentration were significantly higher in large follicles with meiotically competent oocytes compared with those containing meiotically incompetent oocytes. Testosterone levels were increased in PCOS follicles compared with normal patients, with no difference between corresponding sub-groups of follicles with meiotically competent oocytes. The relationship between follicle size and embryo development showed that 14 mm could be a threshold value following rhFSH induction in normal or PCOS women.     

Photoperiodic regulation of body mass and fat reserves in the meadow vole

Short photoperiods reduce both body mass and food intake of male meadow voles. To help determine whether the primary effect of short day lengths is on regulation of some component of body mass or on the control of food consumption, voles housed in a long photoperiod were provided rations equivalent to those consumed by animals in short day lengths. Animals were transferred to a short photoperiod and after either 3 or 6 weeks of food restriction were fed ad lib. Both groups of voles overate after termination of food restriction and achieved seasonally appropriate body masses that were influenced by the duration of short photoperiod treatment. We conclude that short photoperiods gradually alter the regulated level of one or more components of body mass. We tested and failed to support the hypothesis that the subcutaneous fat depot is selectively spared during seasonal weight loss and thereby increases surface insulation. Thus, the proportion of adipose tissue reduction did not differ significantly among the epididymal, retroperitoneal, and inguinal-subcutaneous fat depots of voles housed in short day lengths.     

Localization of mercury in the rat ovary after oral administration of mercuric chloride

The Timm sulphide-silver method was used for histochemical localization of mercury in the ovaries of young rats fed with mercuric chloride. After 12 weeks of mercuric chloride administration test animals showed disturbances in the estrous cycle manifested by the prolongation of diestrous phase. In the ovaries of these rats the deposits of mercury were shown to occur particularly in macrophages of atretic young and mature ovarian follicles and corpora lutea, within granulosa cells of atretic follicles, and in the lutein cells of freshly formed corpora lutea. Follicular fluid and, irregularly, germinal epithelium also showed the ability to mercury accumulation. The ovaries of control animals treated according to the Timm procedure demonstrated negative histochemical reactivity.     

Induction of polycystic ovary by testosterone in immature female rats: Modulation of apoptosis and attenuation of glucose/insulin ratio

Polycystic ovarian syndrome is seen in 5% of fertile aged women. However, there is no satisfactory PCOS model in experimental animals. To induce polycystic ovary phenotype in immature female rats, Wistar rats 21 days of age were injected daily with testosterone propionate 1 mg/100 g body weight dissolved in propylene glycol or propylene glycol for up to 35 days. Seven days of injection with testosterone (T) resulted in the appearance of large cystic follicles and a dramatic accumulation of multi-layer preantral follicles. At 42 days of age puberty in control animals was evident by the appearance of corpora lutea. In contrast in T treated animals no corpora lutea formation was seen even at the age of 56 days. Progesterone in the control animals was elevated at the age of 42 days in contrast with the T treated animals in which progesterone remained low (20% of control). While during 14 days of T injection most of the follicles did not have progressive apoptosis, at 21-35 days of injection (42-56 days of age) the vast majority of follicles became apoptotic. Progressive degeneration of oocytes was evident in T treated animals reaching 70-85% of total oocytes at 21-35 days of T injection compared to 30-40% in control animals. Western blot analysis of ovarian homogenates revealed gradual decrease in Bcl-2 content, evident at 28 and 35 days of T injection compared to control animals. Interestingly, the fasting glucose/insulin ratio was dramatically reduced in T treated animals following 14 days of testosterone treatment compared to controls. Our data suggest that T injection to immature female rats can induce polycystic ovaries, block ovulation and attenuate progesterone production. Moreover, normal/low glucose and high insulin blood levels in the testosterone treated rats raises the possibility that elevated androgens can lead to insulin resistance in this experimental PCOS model.     

Location and developmental regulation of androgen receptor in primate ovary

Locally produced androgens act via granulosa cell androgen receptors to modulate follicular responsiveness to gonadotrophins and thereby contribute to the paracrine regulation of ovarian function. We used quantitative androgen receptor immunocytochemistry to assess androgen receptor distribution in relation to pre-ovulatory follicular development in the common marmoset (Callithrix jacchus), a New World primate that ovulates two to four follicles in each approximately 28 day ovarian cycle. Ovaries from four adult females in the late follicular phase and from four in the luteal phase were fixed in 4% paraformaldehyde and subjected to an immunocytochemical analysis using a polyclonal androgen receptor antibody with detection by a standard avidin-biotin-peroxidase technique for alkaline phosphatase. Specific androgen receptor immunostaining occurred mainly in granulosa cell nuclei, with little or no specific staining in theca, stroma or oocytes. Granulosa cell androgen receptor immunostaining was most abundant in healthy preantral/early antral follicles, being low or absent from pre-ovulatory follicles and corpora lutea. Differences in granulosa cell androgen receptor immunostaining between immature (0.1- 1.0 mm diameter) and pre-ovulatory (> or = 2.0 mm diameter) follicles were quantified using a videodensitometric analysis of grey-scale values. Readings were taken from the granulosa cell layers of 53 immature follicles and 10 pre-ovulatory follicles in late follicular phase ovaries. The average androgen receptor level in granulosa cells of immature follicles proved to be 4.2-fold higher (P < 0.01) than that in granulosa cells of pre-ovulatory follicles. Because other evidence suggests that paracrine androgen action in granulosa cells converts from stimulation to inhibition as follicles mature, we speculate that a development-related reduction in androgen receptor numbers serves to "protect' granulosa cells against the inhibitory action of androgen, thereby promoting pre-ovulatory follicular dominance in primate ovarian cycles.      

Biovularity and "coalescence of primary follicles" in ovaries with mature teratomas

Several theories have been postulated regarding the origin of ovarian teratomas, including incomplete twinning, neoplastic proliferation of sequestered totipotent blastomeres or primordial cells, derepression of totipotent genetic information in the nuclei of somatic cells, and parthenogenetic development of germ cells. At present parthenogenetic development of ova is the most widely accepted theory, primarily because of the presence of a 46 XX karyotype in almost all mature teratomas. However, some authors have raised the possibility of fusion of ova in the mechanism of formation of ovarian teratomas. We report the results of a study on ovarian tissue adjacent to 31 teratomas to assess the frequency of biovularity, which could provide evidence favoring the last theory. On the whole we found biovularity in 26 ovaries of young patients (mean age, 27 years) with variable numbers of biovular follicles ranging from 1 in 4 cases to more than 10 in 2 cases; the number of biovular follicles depended on the quantity of ovarian tissue examined as well as on the total number of ova in the tissue. In multiple occasions 2 ova were included within a single follicle; in 24 ovaries the biovularity was correlated with coalescence of primary follicles characterized morphologically by an ovoid or hourglass-like shape that resulted from cohesion of 2 follicles. As control cases, 30 ovaries of patients with an average age of 28 years were examined (12 removed for endometriosis, 8 for serous cystadenoma, 7 for tubal pregnancy, and 3 for acute salpingo-oophoritis). Only 1 ovary with endometriosis contained a single biovular follicle. The results suggest that ovarian teratoma development may result from fusion of ova in ovaries containing biovularity and phenomena of coalescence of primary follicles.