An ideal research model plays a vital role in studying the pathogenesis of a disease. At present, the most widely used endometrial disease models are cell lines and animal models. As a novel studying model, organoids have already been applied for the study of various diseases, such as disorders related to the liver, small intestine, colon, and pancreas, and have been extended to the endometrium. After a long period of exploration by predecessors, endometrial organoids (EOs) technology has gradually matured and maintained genetic and phenotypic stability after long-term expansion. Compared with cell lines and animal models, EOs have high stability and patient specificity. These not only effectively and veritably reflects the pathophysiology of a disease, but also can be used in preclinical drug screening, combined with patient derived xenografts (PDXs). Indeed, there are still many limitations for EOs. For example, the co-culture system of EOs with stromal cells, immune cell, or vascular cells is not mature, and endometrial cancer organoids have a lower success rate, which should be improved in the future. The investigators predict that EOs will play a significant role in the study of endometrium-related diseases.

Summary Sentence

This review describes the development and potential applications of endometrial organoids.

Aryl hydrocarbon receptor (AhR) is a transcription factor, which can be activated by a plethora of structure-diverse ligands. Historically, AhR is known for its involvements in regulation of metabolism of xenobiotics. However, normal physiological roles of AhR have been defined in other essential biological processes, including vascular growth and function, reproduction, and immunoresponses. In contrast, aberrant expression and activation of the AhR signaling pathway occur in a variety of human diseases, many of which (e.g., preeclampsia, atherosclerosis, and hypertension) could be associated with endothelial dysfunction. Indeed, emerging evidence has shown that either exogenous or endogenous AhR ligands can induce endothelial dysfunction in either an AhR-dependent or AhR-independent manner, possibly reliant on the blood vessel origin (artery and vein) of endothelial cells. Given that the AhR signaling pathway has broad impacts on endothelial and cardiovascular function, AhR ligands, AhR, and their downstream genes could be considered novel therapeutic targets for those endothelial-related diseases. This review will discuss the current knowledge of AhR's mediation on endothelial function and potential mechanisms underlying these actions with a focus on placental endothelial cells.

Summary Sentence

Aryl hydrocarbon receptor critically mediates endothelial function.

Graphical Abstract

Preexisting/pregestational diabetes enhances the risk of birth defects. Several factors have been involved during the implantation process, such as cytokines (granulocyte-macrophage–colonystimulating factor [GM-CSF]). The objective was to evaluate the effects of two levels of diabetes on the redox status of preimplantation embryos during the implantation process to comprehend how both are involved in embryo and fetal viability against maternal diabetes. Female Sprague–Dawley rats received streptozotocin at birth (mild diabetes [MD]) or at adulthood (severe diabetes [SD]) to obtain two experimental diabetes intensities. After confirming the diabetic status, the nondiabetic and diabetic groups were mated around day 110 of life. At gestational day (GD) 21, fetuses were assessed for viability and malformations and ovaries for embryo loss before implantation. Other pregnant nondiabetic and diabetic rats were sacrificed at GD2–4 for maternal and preimplantation embryo oxidative stress markers, maternal serum insulin, uterine fluid GM-CSF, and preimplantation embryo morphological analysis. MD and SD caused abnormal redox levels, lower GM-CSF and insulin levels during the preimplantation period, and embryonic loss before implantation. SD caused lower fetal viability and higher fetal malformation percentages at GD21. The SD dam-derived preimplantation embryos presented lower glutathione levels and higher thiobarbituric acid reactive substances concentration at GD3 and an increased frequency of abnormal preimplantation embryos at GD4. In conclusion, preexisting diabetes leads to complications in the implantation process. Furthermore, maternal oxidative stress and other metabolic changes alter the redox state and morphological structure of preimplantation embryos, contributing to damaged growth and development in late pregnancy.

Summary Sentence

Preexisting diabetes in rats negatively alters maternal cytokine, leading to impaired embryo implantation success. Besides, it damages preimplantation embryo structure and redox status, which contributes to embryofetal loss and fetal abnormalities.

Graphical Abstract

The transcription factor forkhead box L2 (FOXL2) regulates sex differentiation and reproductive function. Elevated levels of this transcription factor have been observed in the diseases of the uterus, such as endometriosis. However, the impact of elevated FOXL2 expression on uterine physiology remains unknown. In order to determine the consequences of altered FOXL2 in the female reproductive axis, we generated mice with over-expression of FOXL2 (FOXL2^OE) by crossing Foxl2^LsL/+ with the Progesterone receptor Pgr^cre model. FOXL2^OE uterus showed severe morphological abnormality including abnormal epithelial stratification, blunted adenogenesis, increased endometrial fibrosis, and disrupted myometrial morphology. In contrast, increasing FOXL2 levels specifically in uterine epithelium by crossing the Foxl2^LsL/+ with the lactoferrin Ltf^icre mice resulted in the eFOXL2^OE mice with uterine epithelial stratification but without defects in endometrial fibrosis and adenogenesis, demonstrating a role of the endometrial stroma in the uterine abnormalities of the FOXL2^OE mice. Transcriptomic analysis of 12 weeks old Pgr^cre and FOXL2^OE uterus at diestrus stage showed multiple signaling pathways related with cellular matrix, wnt/β-catenin, and altered cell cycle. Furthermore, we found FOXL2^OE mice were sterile. The infertility was caused in part by a disruption of the hypophyseal ovarian axis resulting in an anovulatory phenotype. The FOXL2^OE mice failed to show decidual responses during artificial decidualization in ovariectomized mice demonstrating the uterine contribution to the infertility phenotype. These data support that aberrantly increased FOXL2 expressions in the female reproductive tract can disrupt ovarian and uterine functions.

Summary Sentence

FOXL2 overexpression in the uterus induced epithelial stratification, blunted adenogenesis, increased fibrosis, and disrupted myometrium leading to impaired decidual responses.

Graphical Abstract

Development and functions of the ovary rely on appropriate signaling and communication between various ovarian cell types. FOXL2, a transcription factor that plays a key role at different stages of ovarian development, is associated with primary ovarian insufficiency and ovarian cancer as a result of its loss-of-function or mutations. In this study, we investigated the impact of aberrant, constitutive expression of FOXL2 in somatic cells of the ovary. Overexpression of FOXL2 that started during fetal life resulted in defects in nest breakdown and consequent formation of polyovular follicles. Granulosa cell differentiation was impaired and recruitment and differentiation of steroidogenic theca cells was compromised. As a consequence, adult ovaries overexpressing FOXL2 exhibited defects in compartmentalization of granulosa and theca cells, significant decreased steroidogenesis and lack of ovulation. These findings demonstrate that fine-tuned expression of FOXL2 is required for proper folliculogenesis and fertility.

Summary Sentence

Constitutive expression of FOXL2 results in defects in granulosa cell and theca cell differentiation, ultimately impairing folliculogenesis, steroidogenesis and ovulation.

Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies that causes problems in female fertility at the reproductive age. PCOS is a multifactorial disease, with genetic factors playing a crucial role in its development. H19 is a long non-coding RNA (lncRNA) expressed from the maternal chromosome, which is correlated with PCOS. In this study, 115 women suffering from PCOS and 130 healthy women with regular menstrual cycles were recruited as case and control groups, respectively. After the extraction of genomic DNA, the restriction fragment length polymorphism polymerase chain reaction was employed for genotyping of rs2067051G>A and rs3741219T>C. Statistical analysis was done using SPSS package V.22 for Windows. In silico analysis was recruited to determine the effects of SNPs on the secondary structure of gene transcript as well as miRNA binding sites. The obtained data showed that the A allele of rs2067051G>A was associated with the high risk of PCOS (OR = 2.00, 95%CI = 1.38–2.91, P = 0.00). AG and AA genotypes led to a 3.64- and (about) a five-fold increase in the risk of PCOS, respectively (95%CI = 2.02–6.54, P = 0.00, and 95%CI = 1.51–16.52, P = 0.00, respectively). These variants caused a significant increase in the risk of this disorder in all genotype models except in the recessive model. However, no association was found between rs3741219T>C and the increased risk of PCOS, either in the allele or in the genotype models. According to the findings, rs2067051G>A is associated with an increased risk of PCOS in the Iranian population.

Maternal aging affects various aspects of oocytes' physiology, including the functionality of their nuclear apparatus and mitochondria. In the present paper, we wished to investigate whether advanced reproductive age impacts oocytes' ability to generate proper Ca²⁺ oscillations in response to monospermic fertilization. We examined three different mouse strains/crosses: inbred C57BL/6Tar, outbred Tar:SWISS, and hybrid F1 (C57BL/6Tar × CBA/Tar). The females were either 2–4 months old (young) or 13–16 months old (aged). We observed that the Ca²⁺ oscillatory pattern is altered in a strain-dependent manner and changes were more profound in aged C57BL/6Tar and F1 than in aged Tar:SWISS oocytes. We also showed that maternal aging differently affects the size of Ca²⁺ store and expression of Itpr1, Atp2a2, Erp44, and Pdia3 genes involved in Ca²⁺ homeostasis in oocytes of C57BL/6Tar, Tar:SWISS, and F1 genetic background, which may explain partially the differences in the extent of age-dependent changes in the Ca²⁺ oscillations in those oocytes. Maternal aging did not have any visible impact on the distribution of the ER cisterns in oocytes of all three genetic types. Finally, we showed that maternal aging alters the timing of the first embryonic interphase onset and that this timing correlates in C57BL/6Tar and Tar:SWISS oocytes with the frequency of fertilization-induced Ca²⁺ oscillations. Our results indicate that extreme caution is required when conclusions about oocyte/embryo physiological response to aging are made and complement an increasing amount of evidence that mammalian (including human) susceptibility to aging differs greatly depending on the genetic background of the individual.

Summary Sentence

The impact of maternal aging on Ca²⁺ homeostasis in fertilized mouse oocytes varies depending on the genetic origin of females.

Graphical Abstract

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC–MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins was identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition - and hence independent of transcription - were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell–cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

Summary sentence

For oocyte maturation, selective functional classes of proteins are dependent on newly synthesized messenger RNAs.

The two-way communication between the mother and the fetus is accomplished by immune cells. CD8+ T cells of normal pregnant (NP) women express progesterone receptor (PR). Binding of PR to progesterone (P) and the production of progesterone-induced blocking factor (PIBF) can aid immune escape, which is an important factor in the maternal immune response. We detected the proportion of CD8+ T cells and the expression of the surface costimulatory molecules BTLA, TIGIT, ICOS, and PD-1 in peripheral blood and decidual tissues of women with unexplained recurrent spontaneous abortion (URSA) and in NP women. All patients were at 8 -10 weeks of gestation. The results showed that there was no change in the proportions of CD8+ T cells in peripheral blood and decidual tissues of URSA patients compared to those of NP women. In peripheral blood, compared with the NP group, the URSA group showed decreased expression of BTLA + CD8+ T cells and the difference was statistically significant, but there was no difference between the groups in terms of TIGIT + CD8+, PD-1 + CD8+, and ICOS + CD8+ T cells. There was no change in the levels of TIGIT + CD8+, PD-1 + CD8+, ICOS + CD8+, and BTLA + CD8+ T cells in decidual tissue. These data confirm that the number of CD8+ T cells in peripheral blood and decidual tissue is not the main factor leading to the pathogenesis of URSA, and other immune cells may play an important role in URSA, but this hypothesis needs further exploration and research.

The emerging paradigm in the immunology of pregnancy is that implantation of conceptuses does not progress in an immunologically suppressed environment. Rather, the endometrium undergoes a controlled inflammatory response during implantation as trophectoderm of elongating and implanting pig conceptuses secrete the pro-inflammatory cytokine interferon gamma (IFNG). Results of this study with pigs revealed: (1) accumulation of immune cells and apoptosis of stromal cells within the endometrium at sites of implantation during the period of IFNG secretion by conceptuses; (2) accumulation of proliferating cell nuclear antigen (PCNA)-positive T cells within the endometrium at sites of implantation; (3) significant increases in expression of T cell co-signaling receptors including programmed cell death 1 (PDCD1), CD28, cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and inducible T cell co-stimulator (ICOS), as well as chemokines CXCL9, 10, and 11 within the endometrium at sites of implantation; (4) significant increases in T cell co-signaling receptors, PDCD1 and ICOS, and chemokine CXCL9 in the endometrium of cyclic gilts infused with IFNG; and (5) identification of CD4⁺ (22.59%) as the major T cell subpopulation, with minor subpopulations of CD8⁺ (1.38%), CD4⁺CD25⁺ (1.08%), and CD4⁺CD8⁺ (0.61%) T cells within the endometrium at sites of implantation. Our results provide new insights into the immunology of implantation to suggest that trophectoderm cells of pigs secrete IFNG to recruit various subpopulations of T cells to the endometrium to contribute to a controlled inflammatory environment that supports the active breakdown and restructuring of the endometrium in response to implantation of the conceptus.

Summary Sentence

Pig conceptus IFNG recruits T cells to the endometrium to contribute to a tightly controlled inflammatory environment that supports the active breakdown and restructuring of the endometrium in response to implantation of the conceptus.

Although sexual transmission of Zika virus (ZIKV) is well-documented, the viral reservoir(s) in the male reproductive tract remains uncertain in humans and immune-intact animal models. We evaluated the presence of ZIKV in a rhesus macaque pilot study to determine persistence in semen, assess the impact of infection on sperm functional characteristics, and define the viral reservoir in the male reproductive tract. Five adult male rhesus monkeys were inoculated with 10⁵ PFU of Asian-lineage ZIKV isolate PRVABC59, and two males were inoculated with the same dose of African-lineage ZIKV DAKAR41524. Viremia and viral RNA (vRNA) shedding in semen were monitored, and a cohort of animals were necropsied for tissue collection to assess tissue vRNA burden and histopathology. All animals exhibited viremia for limited periods (1–11 days); duration of shedding did not differ significantly between viral isolates. There were sporadic low levels of vRNA in the semen from some, but not all animals. Viral RNA levels in reproductive tract tissues were also modest and present in the epididymis in three of five cases, one case in the vas deferens, but not detected in testis, seminal vesicles or prostate. ZIKV infection did not impact semen motility parameters as assessed by computer-assisted sperm analysis. Despite some evidence of prolonged ZIKV RNA shedding in human semen and high tropism of ZIKV for male reproductive tract tissues in mice deficient in Type 1 interferon signaling, in the rhesus macaques assessed in this pilot study, we did not consistently find ZIKV RNA in the male reproductive tract.

Summary Sentence

Male rhesus monkeys, evaluated 1–6 weeks following subcutaneous Zika virus inoculation, have sporadic presence of viral RNA in the epididymis and semen.

The Arctic aphids live briefly and must breed quickly to survive. Shortened life cycle, with only two generations: the stem mother and sexuales—oviparous females and males is an adaptation for optimal use of the short breeding period, which lasts from late July to the end of August. Using Acyrthosiphon svalbardicum, an endemic High Arctic aphid species, we describe the structure of the reproductive system of sexual morphs and compare with its temperate counterparts, in particular the model organism the pea aphid Acyrthosiphon pisum. Generally, the histological composition and ultrastructure of reproductive system of sexuales of A. svalbardicum is broadly similar to the reproductive systems described already in other species of aphids. The unique characters include in both oviparous females and males an enormous layer of the fat body, adhering to the structures of the internal reproductive system. The greatly enlarged accessory glands of males accumulate a heterogenous secretion composed of irregularly organized bunches of spicule-like structures of high electron density embedded in fine and coarse granular material. This material, unknown among temperate counterparts of A. svalbardicum, during mating is transported from the accessory glands of the male to its ejaculatory duct, where it is mixed with the ejaculate, and then is transferred to the spermatheca of the oviparous female.

Summary Sentence

In sexuales of an endemic species Acyrthosiphon svalbardicum, unique characters of the reproductive system include an enormous layer of the fat body and the presence of the spicule-like structures, produced by enlarged accessory glands of male.

Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed that BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in polyovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in polyovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at preantral stages, with formation of numerous structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in nonrodent polyovulatory species may be as important as that in mono-ovulatory species.

Summary sentence

Examination of BMP15-edited cloned pigs indicated that loss of function of BMP15 severely affects porcine female fertility, mainly because of the inhibition of follicular development into the antral stage.

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared with controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, luteinizing hormone (LH)-stimulated cyclic adenosine monophosphate (cAMP) generation, and LH receptor messenger RNA (mRNA) expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E₂ in prepubertal gilts and PGF_2alpha metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as human chorionic gonadotropin- and forskolin-induced cAMP were also affected. In addition, altrenogest downregulated CYP17A1 mRNA in the prepubertal theca layer and PGF_2alpha synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

Summary Sentence

Sexual maturity and altrenogest change endocrine milieu and molecular regulations governing folliculogenesis in gilts.

Women with polycystic ovary syndrome (PCOS) are characterized by endocrine disorders accompanied by a decline in oocyte quality. In this study, we generated a PCOS mice model by hypodermic injection of dehydroepiandrosterone, and metformin was used as a positive control drug to study the effect of pachymic acid (PA) on endocrine and oocyte quality in PCOS mice. Compared with the model group, the mice treated with PA showed the following changes (slower weight gain, improved abnormal metabolism; increased development potential of GV oocytes, reduced number of abnormal MII oocytes, and damaged embryos; lower expression of ovarian-related genes in ovarian tissue and pro-inflammatory cytokines in adipose tissue). All these aspects show similar effects on metformin. Most notably, PA is superior to metformin in improving inflammation of adipose tissue and mitochondrial abnormalities. It is suggested that PA has the similar effect with metformin, which can improve the endocrine environment and oocyte quality of PCOS mice. These findings suggest that PA has the similar effect with metformin, which can improve the endocrine environment and oocyte quality of PCOS mice.

Graphical Abstract

Deciphering mechanisms of oocyte development in the fish ovary still remain challenging, and a comprehensive overview of this process at the level of the organ is still needed. The recent development of optical tissue clearing methods has tremendously boosted the three-dimensional (3D) imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the nontoxic solvent-based ethyl cinnamate (ECi) method for efficient clearing and the Clear Unobstructed Brain Imaging Cocktails and Computational (CUBIC) method to enhance lipid removal and reduce nonspecific staining. The methyl green fluorescent dye was used to stain nuclei and delineate the follicular structures that include oocytes. Using this procedure (named CUBIC-ECi [C-ECi]), ovaries of both medaka and trout could be imaged in 3D and follicles analyzed. To our knowledge, this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-ECi method thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further developmental and morphological studies.

Summary Sentence

A modified ethylcinnamate-based clearing method allowing 3D imaging of fish ovary and analyzing follicular content.

Graphical Abstract

Sex steroids regulate insulin sensitivity and glucose metabolism. We had characterized a lean type 2 diabetes (T2D) rat model using gestational low-protein (LP) diet programming. Our objective was to identify if endocrine dysfunction leading to decreased sex hormone levels will precede the development of T2D and if steroid replacement will prevent the onset of the disease. Pregnant rats were fed control or isocaloric LP diet from gestational day 4 until delivery. Normal diet was given to all mothers after delivery and to pups after weaning. LP offspring developed glucose intolerance and insulin resistance at 4 months. We measured sex steroid hormone profiles and expression of key genes involved in steroidogenesis in testis and ovary. Furthermore, one-month old rats were implanted with 90-day slow release T and E2 pellets for males and females, respectively. Glucose tolerance test (GTT) and euglycemic hyperinsulinemic clamp was performed at 4 months. LP-programmed T2D males had low T levels and females had low E2 levels due to dysregulated gene expression during steroidogenesis in gonads. GTT and euglycemic hyperinsulinemic clamp showed that LP males and females were glucose intolerant and insulin resistant; however, steroid supplementation prevented the onset of glucose intolerance and insulin resistance. Rats that developed T2D by LP programming have compromised gonadal steroidogenesis leading to low T and E2 in males and females, respectively. Sex steroid supplementation prevented the onset of glucose intolerance and insulin resistance indicating low sex steroid levels could cause compromised glucose metabolism ultimately leading to T2D.

Summary Sentence

Low-protein-programmed type 2 diabetes is mediated by testosterone and estradiol in males and females, respectively, in rats.

Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in nonobstructive azoospermia patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis, or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis, or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.

Summary sentence

AK1 is required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.

Sirolimus, also known as rapamycin, and its closely related rapamycin analog (rapalog) Everolimus inhibit “mammalian target of rapamycin complex 1” (mTORC1), whose activity is required for spermatogenesis. Everolimus is Food and Drug Administration approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P>60) mice received daily injections of sirolimus or Everolimus for 30 days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus ∼6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration and dose dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring.

Summary Sentence

Treatments of juvenile and adult mice with rapamycin and its analog (rapalog) Everolimus cause reproductive deficits, but males are able to sire offspring following cessation of treatment.