Penelope L. Nayudu

Impact of Carbohydrate Heterogeneity in Function of Follicle-Stimulating Hormone: Studies Derived from in Vitro and in Vivo Models

Abstract Carbohydrates attached to the protein core of glycoprotein hormones influence a number of intracellular and extracellular processes. As with other members of the glycoprotein hormone family, FSH is produced and released as an array of isoforms that differ from each other in the structure of their oligosaccharide attachments. In this review, we discuss how carbohydrate heterogeneity can impact on FSH action in different in vitro and in vivo systems. We present evidence for diverse effects of distinct charge isoforms at the target cell level, including differential and unique effects on various end responses, and discuss how the use of multiple cell-type assays has allowed identification of some specific effects of FSH isoforms on different cell populations and follicle compartments as well as oocyte maturation. Finally, we discuss recent information on the ability of naturally occurring and laboratory manufactured FSH isoforms to evoke particular effects on granulosa cell function and ovarian follicular maturation in vivo. Such studies have provided evidence that the type(s) of FSH signal delivered may in fact regulate distinct biological outcomes irrespective or in addition to outcomes dictated solely by clearance rate differences.

Luteinizing hormone has a stage-limited effect on preantral follicle development in vitro.

Abstract Although it is known that LH receptors are present from the time of thecal differentiation, the role of LH during early follicle development is not yet clear. The effect of LH on preantral follicle development has therefore been investigated in vitro using a culture system that supports the development of intact follicles. We have previously shown that although preantral follicles 150 μm in diameter (2–3 granulosa cell layers) do not require LH to proceed through antral development, smaller follicles (1–2 granulosa cell layers, 85–110 μm in diameter) do not develop beyond the large preantral stage in the presence of only FSH and 5% mouse serum. Follicles of this size were therefore used to determine the effects of LH and serum on their development in vitro. The results showed that although FSH must be continuously present, a low concentration of LH together with a slight increase in serum concentration was necessary, specifically during the primary stage of follicle development (from 85 μm in diameter until the follicles had reached 150 μm in diameter) to induce the capacity for subsequent LH-independent rapid growth and antral development. The in vitro development of maturable oocytes with normal spindle and chromatin morphology was also supported. These results indicate that LH probably induces changes in the early differentiating thecal cells, which are critical for the completion of subsequent follicular and oocyte development.

Intact follicle culture: what it can tell us aabout the roles of FSH glycoforms during follicle development

An important limiting factor in assisted reproduction treatment success rates is oocyte quality. In spite of improved results through several important innovations, the pregnancy rate per collected oocyte remains far too low. In order to improve this situation, it is necessary to learn more about fundamental factors modulating follicular development patterns. FSH is known to be the driving force for follicle development, but it is not yet understood how its multifarious functions are controlled and modulated. Evidence is accumulating that FSH glycoforms may be the key to this mystery. Intact follicle culture is a useful tool for the clarification of the actions of the different isoforms because the follicle unit is maintained and allowed to develop through several critical stages. Additionally important is the availability of the oocyte for functional evaluation. Because of these features, relationships can be uncovered that are not revealed with single cell test systems. The results so far obtained with this system suggest that follicle development pattern and oocyte quality is strongly influenced by FSH glycoform range, and that the requirements of the follicle may shift during progress through different stages of development. More studies are required, but these findings already suggest that the physiological shifts of circulating FSH glycoforms may indeed be important, and that attention should be paid to the glycoform distribution of exogenously applied FSH.

Progress Toward Understanding Follicle Development in Vitro : Appearances Are Not Deceiving

The interactive factors that influence the developmental progress of a follicle and determine whether it will progress to ovulation or toward atresia, are highly complex. In vitro models are being developed that are intended to provide a simplified environment to facilitate understanding of the dynamics of the processes involved. The purpose of this overview is to evaluate progress to date and to focus attention on issues that need more careful consideration to improve the usefulness of the models. Basically, two approaches exist. One, attached follicle culture, employs either enzyme-digested or mechanically harvested follicles depending on the method but allows attachment of the follicles to the culture surface. This produces a rounded or flattened structure (depending on culture conditions) that is no longer an intact follicle. During this culture, the cells reorganize themselves, some remaining in contact with the oocyte and others attaching to the culture surface and proliferating. The other approach, intact 3-dimensional follicle culture, employs mechanically dissected preantral follicles that are cultured as free-floating intact structures. Intact follicle culture emulates the in vivo developmental pattern of the follicle more closely than a non-intact structure can, and thereby provides a favorable model to investigate the interaction between hormonal and paracrine factors in the development of the follicle in isolation from systemic effects. For example, intact follicle culture has begun to be used to investigate the local effects of several different steroids. In addition, the local effects of inhibin, activin, and follistatin and their interactions with locally produced growth factors and steroids as well as synergy with gonadotrophins are beginning to be investigated. In our laboratory, the focus is on the roles of gonadotrophins at different stages of follicle development, particularly the effect of FSH isoforms in modulating follicle development in vitro. Finally, an important issue that urgently needs to be addressed, for future studies of in vitro follicle development, is the rationalization and standardization of follicle culture conditions.

Congenitally caused fused labia in the common marmoset (Callithrix jacchus)

Abstract: In this paper, the occurrence of an external genital abnormality in female marmoset monkeys (fused labia) is discussed. This malformation was detected, for the first time, in a group of animals at the German Primate Center (GPC), Goettingen. The malformed vulva was completely sealed except for an opening of 1.5–2.5 mm around the urethra sufficient for urination. Because of this defect the animals were not able to copulate. As a consequence, the affected females were functionally infertile although they had a normal genital tract and a regular cycle. This vulvar abnormality was found in 12 females, offspring of 10 pairs in which either one or both came to the German Primate Center from two genetically related colonies in Munich, Germany, and one colony in Basel, Switzerland. The abnormality appeared to be recessive and inheritable from either parent. In pairs in which both animals were from one of the mentioned colonies, 45% of the female offspring were affected. In pairs where only one partner came from these colonies, 26% of female offspring had the malformation. These results indicate that avoidance of inbreeding, which is frequently performed in primate colonies, may reduce, but not eliminate the expression of abnormalities of genetic origin. Therefore selective breeding is required, and, in colonies where these recessive mutations are widespread, the development of genetic screening tests would be advantageous.

Embryonic Development after Follicle Culture Is Influenced by Follicle-Stimulating Hormone Isoelectric Point Range

Abstract We evaluated the effects of follicular exposure in vitro to either of two mutually exclusive isoforms of FSH (least acidic and acid) on the subsequent capacity of oocytes for embryonic development. The effects of dose and follicle culture duration were examined. At the threshold dose (that required to produce antra) and at one subthreshold dose, the major difference between the two isoform fractions was the timing and effectiveness of acquisition of two-cell embryonic developmental capacity. With the least-acidic fraction, the highest rate of two-cell development (≈80%) occurred after 3 days of follicle culture only at the threshold dose (2.5 ng/ml). With the acid fraction, the highest two-cell rate (≈60%) occurred after 5 days of culture but at equivalent rates over a range of doses between 10 ng/ml and 100 ng/ml (threshold dose was 50 ng/ml). At threshold dose or below, the capacity for two-cell embryo production appeared not to be influenced by antral status for either isoform. At above threshold doses, the least-acidic fraction induced an increasing proportion of antral follicles with increasing dose, but this increase was associated with a progressive decrease in embryo production. This relationship was more extreme after longer culture and was due to degeneration of the cumulus-oocyte complex associated with apparently increased differentiation of the mural granulosa cells. The acid fraction was by comparison less bioactive and insensitive to overdosing. The broader isoform mix of the unfractionated FSH provided a measure of protection against overdosing characteristic of the acid fraction while retaining the capacity of the least-acidic fraction to induce antral formation at a low dose.

Variations of chromatin, tubulin and actin structures in primate oocytes arrested during in vitro maturation and fertilization—what is this telling us about the relationships between cytoskeletal and chromatin meiotic defects?

A nonhuman primate model was applied to investigate the relationships between variations in the organization of microtubules, microfilaments, and chromatin in metaphase I and metaphase II oocytes. Marmoset oocytes were subjected to in vitro maturation and coincubation with sperm. Oocytes which failed to cleave were investigated for chromatin, tubulin, and actin using Hoechst 33258, fluorescein isothiocyanate (FITC)-labeled alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. Spindles were categorized according to size, shape and microtubule organization: normal, large, multipolar, disorganized, absent spindle, and spindles with broad poles. Actin caps were categorized as: normal, small, split, and disorganized. Chromosomal condensation and alignment were described as normal or abnormal. Improper chromosomal condensation was associated with both abnormal microfilament and microtubule arrangement. This was further associated with abnormal actin organization, disorientation and late stabilization of microtubules, but not related to abnormal organization of spindle poles. Chromosomal misalignment was associated with disorientation and late stabilization of tubulin, but not to broad spindle pole. Additionally, abnormal actin polarization appeared not to be related to abnormal spindle poles. The model system presented in this study could be used as an experimental platform for studying the contribution of different factors to the exactness of late meiotic events in primate oocytes. The present study provides basic information on spindle, chromosome, and actin normal and abnormal organization, which can be observed in in vitro matured, but failed to cleave primate oocytes.

The Common Marmoset (Callithrix jacchus) Has Two Very Similar Semenogelin Genes as the Result of Gene Conversion

Abstract The semen coagulum proteins have undergone substantial structural changes during evolution. In primates, these seminal vesicle-secreted proteins are known as semenogelin I (SEMG1) and semenogelin II (SEMG2). Previous studies on the common marmoset (Callithrix jacchus) showed that ejaculated semen from this New World monkey contains semenogelin, but it remained unclear whether it carries both genes or only SEMG1 and no SEMG2, like the closely related cotton-top tamarin (Saguinus oedipus). In this study we show that there are two genes, both expressed in the seminal vesicles. Surprisingly, the genes show an almost perfect sequence identity in a region of 1.25 kb, encompassing nearly half of the genes and containing exon 1, intron 1, and the first 0.9 kb of exon 2. The underlying molecular mechanism is most likely gene conversion, and a phylogenetic analysis suggests that SEMG1 is the most probable donor gene. The marmoset SEMG1 in this report differs from a previously reported cDNA by a lack of nucleotides encoding one repeat of 60 amino acids, suggesting that marmoset SEMG1 displays allelic size variation. This is similar to what was recently demonstrated in humans, but in marmosets the polymorphism was generated by a repeat duplication, whereas in humans it was a deletion. Together, these studies shed new light on the evolution of semenogelins and the mechanisms that have generated the structural diversity of semen coagulum proteins.

Critical estradiol dose optimization for oocyte in vitro maturation in the common marmoset

The aim of the present study was to critically evaluate the effect of different concentrations of estradiol (E2) during IVM of common marmoset (Callithrix jacchus) oocytes from antral follicles. The doses tested were 0, 0.1, 1, or 10 μg/mL E2 (referred to as 0 E2, 0.1 E2, 1 E2, and 10 E2 groups). After a preincubation, the concentration of E2 in IVM drops under oil was approximately 20% of the amount added (0.02; 0.2 and 1.9 μg/mL, respectively) because of absorption into the oil. Oocyte progression to metaphase II was significantly higher in the 0.1 E2 group than that in the absence of E2. With progressively higher doses, the maturation rate tended to decrease suggesting an overdose effect. Furthermore, the total first cleavage rate was significantly higher in the 0.1 E2 group than that in the 0 E2 group and decreased progressively with further increases in E2 concentration, with the 10 E2 group showing the same low rate as without E2. The oocytes which failed to cleave, after maturation in 10 E2, showed obvious signs of overdose with the highest rates of degeneration and abnormal spindle form, and an absence of embryo progression. In contrast to these obvious negative effects on the oocyte, 10 E2 was the only group in which a significant increase in radial cumulus expansion was observed. The concentration 0.1 E2, which is 10 times lower than the most commonly used E2 dose, produced the best results in all oocyte factors evaluated. These results represent the first study for a primate species showing a strong positive effect of E2 on oocyte maturation and embryo development, but only at the optimal concentration, and emphasize the critical limits of the optimal concentration range.

Abnormal in vitro development of ovarian follicles explanted from mice exposed to tetrachlorvinphos.

A system of mouse ovarian follicle culture in which follicles can be grown from a preantral stage of development through antral formation has been developed and modified recently by Nayudu and colleagues. Follicles have been shown to grow in this culture system at a relatively constant rate and show responsiveness to LH at the end of the culture by ovulation of mature oocytes. Reported here are the distinctly different in vitro growth patterns of follicles explanted from 22- to 24-day-old mice during a period when the colony was being treated for skin parasites with tetrachlorvinphos (TCVP) (Rabond). There is to date no information on the effects of this compound on the mammalian female reproductive system. For follicles from the TCVP treated group, the duration of growth as intact follicles was markedly reduced in comparison to mice of the same strain and source not treated with TCVP. In the treated group, premature termination of follicular growth was also associated with the spontaneous expulsion of oocytes with immature nuclei and without cumulus cells. For those follicles from treated mice that did remain in culture until the day luteinizing hormone was given, the ovulatory response was poor and the maturation response of the oocytes was low in comparison with the follicles from untreated mice. The effect of the treatment on the follicles was further characterized by obvious differences in the patterns of growth. Follicles in the untreated group grew in a linear pattern at around 25 microns/day; a single phase, fast pattern for the whole culture period.(ABSTRACT TRUNCATED AT 250 WORDS)