Many genes important for reproductive performance are shared by both sexes. However, fecundity indices are primarily based on female parameters such as litter size. We examined a fertility mouse line (FL2), which has a considerably increased number of offspring and a total litter weight of 180% compared to a randomly bred control line (Ctrl) after more than 170 generations of breeding. In the present study, we investigated whether there might be a parallel evolution in males after more than 40 years of breeding in this outbred mouse model. Males of the fertility mouse line FL2 showed reduced sperm motility performance in a 5 h thermal stress experiment and reduced birth rate in the outbred mouse line. Transcriptional analysis of the FL2 testis showed the differential expression of genes associated with steroid metabolic processes (Cyp1b1, Cyp19a1, Hsd3b6, and Cyp21a1) and female fecundity (Gdf9), accompanied by 150% elevated serum progesterone levels in the FL2 males. Cluster analysis revealed the downregulation of genes of the kallikrein-related peptidases (KLK) cluster located on chromosome 7 in addition to alterations in gene expression with serine peptidase activity, e.g., angiotensinogen (Agt), of the renin-angiotensin system essential for ovulation. Although a majority of functional annotations map to female reproduction and ovulation, these genes are differentially expressed in FL2 testis. These data indicate that selection for primary female traits of increased litter size not only affects sperm characteristics but also manifests as transcriptional alterations of the male side likely with direct long-term consequences for the reproductive performance of the mouse line.

The development and maturation of ovarian follicles is a complex and highly regulated process, which is essential for successful ovulation. During recent decades, several mouse models provided insights into the regulation of folliculogenesis. In contrast to the commonly used transgenic or knockout mouse models, the Dummerstorf high-fertility mouse line 1 (FL1) is a worldwide unique selection experiment for increased female reproductive performance and extraordinary high fertility. Interactions of cycle-related alterations of parameters of the hypothalamic pituitary gonadal axis and molecular factors in the ovary lead to improved follicular development and therefore increased ovulation rates in FL1 mice. FL1 females almost doubled the number of ovulated oocytes compared to the unselected control mouse line. To gain insights into the cellular mechanisms leading to the high fertility phenotype we used granulosa cells isolated from antral follicles for mRNA sequencing. Based on the results of the transcriptome analysis we additionally measured hormones and growth factors associated with follicular development to complement the picture of how the signaling pathways are regulated. While IGF1 levels are decreased in FL1 mice in estrus, we found no differences in insulin, prolactin and oxytocin levels in FL1 mice compared to the control line. The results of the mRNA sequencing approach revealed that the actions of insulin, prolactin and oxytocin are restricted local to the granulosa cells, since hormonal receptor expression is differentially regulated in FL1 mice. Additionally, numerous genes, which are involved in important gonadotropin, apoptotic and metabolic signaling pathways in granulosa cells, are differentially regulated in granulosa cells of FL1 mice.We showed that an overlap of different signaling pathways reflects the crosstalk between gonadotropin and growth factor signaling pathways, follicular atresia in FL1 mice is decreased due to improved granulosa cell survival and by improving the efficiency of intracellular signaling, glucose metabolism and signal transduction, FL1 mice have several advantages in reproductive performance and therefore increased the ovulation rate. Therefore, this worldwide unique high fertility model can provide new insights into different factors leading to improved follicular development and has the potential to improve our understanding of high fertility.

In recent decades, fertility traits in humans as well as in farm animals have decreased worldwide. As such, it is imperative to know more about the genetics and physiology of increased or high fertility. However, most of the current animal models with reproductive phenotypes describe lower fertility or even infertility (around 99%). The “Dummerstorf high-fertility lines” (FL1 and FL2) are two unique mouse lines selected for higher reproductive performances, more specifically for higher number of pups per litter. We recently described how those superfertile mice managed to increase their reproductive phenotype by doubling the ovulation rate and consequently the litter size compared to the unselected mice of the same founder population. FLs show an unusual estrous cycle length and atypical levels of hormones that link reproduction and metabolism, such as insulin in FL1 and leptin in FL2. Moreover, we described that their higher ovulation rate is mostly due to a higher quality of their oocytes rather than their sheer quantity, as they are characterized by a higher quantity of high-quality oocytes in antral follicles, but the quantity of follicles per ovary is not dissimilar compared to the control. In the present study, we aimed to analyze the lipid composition of the fertility lines from plasma to the gonads, as they can connect the higher reproductive performances with their metabolic atypicalities. As such, we analyzed the fat content of FLs and fatty acid composition in plasma, liver, fat, oocytes of different quality, and granulosa cells. We demonstrated that those mice show higher body weight and increased body fat content, but at the same time, they manage to decrease the lipid content in the ovarian fat compared to the abdominal fat, which could contribute to explaining their ovarian quality. In addition, we illustrate the differences in fatty acid composition in those tissues, especially a lower level of saturated fatty acids in plasma and a different lipid microenvironment of the ovary. Our ongoing and future research may be informative for farm animal biology as well as human reproductive medicine, mostly with cases that present characteristics of lower fertility that could be reversed following the way-of-managing of Dummerstorf high-fertility lines.

Mouse models of decreased fertility mainly support scientific knowledge in the field of reproductive biology. In this study, we changed the perspective, using Dummerstorf high‐fertility mouse lines FL1 and FL2 selected for increased reproductive performances that doubled the amount of ovulated oocytes per cycle and the number of offspring per litter compared to an unselected control line (founder population, FZTDU, ctrl). After recent observations, both fertility lines seem to show a lower pregnancy rate compared to ctrl together with an atypical reproductive cycle. We analysed the estrous cycle of those mice, but also plasma levels of insulin, glucagon, leptin and ghrelin that, when irregular, may have an impact on the reproductive cycle length by modifying the GnRH release. We included females of another independent line (DU6P), selected from the same founder population but independently of fertility traits, as an additional model of decreased pregnancy rate. We aim to evaluate if do they use a similar mechanism associated in the regulation of the estrous cycle or implicated in altered pregnancy mechanism compared to control, because they show a similar situation as FL2 line does, even without an increase in fertility parameters. We speculate that FLs’ estrous cycle undergoes changes during the selection period and aim to demonstrate that some hormonal dysfunctions link with altered reproductive cycle, dampened pregnancy rates and reduced first delivery rates mostly in FL2, but also with higher‐fertility phenotype rather than lower in both FLs.

Factors of high fertility are poorly described. The majority of transgenic or knockout models with a reproductive phenotype are subfertile or infertile phenotypes. Few genotypes have been linked to improved reproductive performance (0.2%) or increased litter size (1%). In this study, we used a unique mouse model, fertility line FL1, selected for ‘high fertility’ for more than 170 generations. This strain has almost doubled the number of littermates as well as their total birth weight accompanied by an elevated ovulation rate and increased numbers of corpora lutea compared to a randomly mated and unselected control line (Ctrl). Here, we investigate whether the gonadal tissue of FL1 males are affected by ‘co-evolution’ after more than 40 years of female-focused selection. Using microarrays, we analysed the testicular transcriptome of the FL1 and Ctrl mice. These data were also compared with previously published female gonadal transcriptional alterations. We detected alterations in testicular gene expression, which are partly associated with female reproductive performance. Thus, female-focused selection for litter size has not only affected the female side, but also has been manifested in transcriptional alterations on the male gonadal organ. This suggests consequences for the entire mouse lines in the long run and emphasizes the perspective of inevitably considering both genders about mechanisms of high fertility.

Dummerstorf fertility lines FL1 and FL2 represent two models of enhanced fertility characterized by the doubling of the litter size compared with an unselected control population (ctrl line, Dummerstorf FztDU). Both biodiverse FLs managed to reach this goal by increasing the ovulation rate per cycle, even showing decreased pregnancy rate and irregular oestrous cycle and metabolic hormone levels, compared with ctrl. The aim of the present study was to analyse oocytes in terms of quality and quantity by comparing the entire pool of oocytes per ovary, with those from the antral follicles within the same animal. We performed Brilliant Cresyl Blue staining as a non‐invasive marker of oocyte quality in combination with an analysis of additional morphological indicators, e.g. cytoplasm clarity, cumulus cell layers, nuclear anatomy, size and shape. We compared our fertility lines with the unselected control population and with another independent line selected from the same founder population, showing lower litter size (DU6P). Our results suggest that fertility lines show decreased number of oocytes per ovary compared with DU6P but increased number of high‐quality oocytes before ovulation. Hence, the raise in the ovulation rate and litter size of those super fertile mouse lines are not associated with an increased number of oocytes per ovary but rather with an increased number of higher quality fertilizable oocytes per cycle. In addition, the most conspicuous method to acquire oocytes with the highest quality in our lines is to assess their morphology, rather than their status after staining. All these discoveries together may be of fundamental importance for further studies in livestock farm animals showing some similar characteristics, e.g. irregular cycle or hormonal misbalances, to improve production while lowering costs, and in humans to increase the possibilities of successful pregnancies for couples undergoing in vitro fertilization (IVF).